Anti-infective therapy

ABSTRACT

DNA isolates coding for human DNase and methods of obtaining such DNA are provided, together with expression systems for recombinant production of human DNase useful in therapeutic or diagnostic compositions.

[0001] This invention is a continuation-in-part of U.S. Ser. No.07/289,958 filed Dec. 23, 1988. This invention relates to new methodsfor making deoxyribonuclease (DNase), especially human DNase, and tonucleic acid encoding DNase.

[0002] DNase is a phosphodiesterase capable of hydrolyzingpolydeoxyribonucleic acid. It acts to extensively and non-specificallydegrade DNA and in this regard is distinguished from the relativelylimited and sequence-specific restriction endonucleases. This inventionis concerned principally with DNase I and II. DNase I has a pH optimumnear neutrality, an obligatory requirement for divalent cations, andproduces 5′-phosphate nucleotides on hydrolysis of DNA. DNase IIexhibits an acid pH optimum, can be activated by divalent cations andproduces 3′-phosphate nucleotides on hydrolysis of DNA. Multiplemolecular forms of DNase I and II also are known.

[0003] DNase from various species has been purified to varying degree.Bovine DNase A, B, C, and D was purified and completely sequenced asearly as 1973 (Liao et al., J. Biol. Chem. 248:1489 [1973]; Salnikow etal., J. Biol. Chem. 248:1499 [1973]; Liao et al., J. Biol. Chem.249:2354 [1973]). Porcine and ovine DNase have been purified and fullysequenced (Paudel et al., J. Biol. Chem. 261:16006 [1986] and Paudel etal., J. Biol. Chem. 261:16012 [1986]). Human urinary DNase was reportedto have been purified to electrophoretically homogeneous state and theN-terminal amino acid observed to be leucine; no other sequence wasreported (Ito et al., J. Biochem. 95:1399 [1984]; see also Funakoshi etal., J. Biochem. 82:1771 [1977]; Murai et al., Biochim. et Biophys. Acta517:186 [1978] and Wroblewski et al., P.S.E.B.M. 74:443 [1950]).

[0004] Notwithstanding that full sequence information for a mammalianDNase first became known in 1973, only recently has a report appeared ofan attempt to clone and express this class of enzymes. Shields et al.describe the expression cloning of part of the gene for bovine DNase Iand expression of a fusion product in E. coli which was biologically andimmunologically active (Biochem. Soc. Trans. 16:195 [1988]). The DNaseproduct of Shields et al., however, was toxic to the host cells andcould only be obtained by the use of an inducible promoter. Furthermore,great difficulty was encountered in attempts to isolate plasmid DNA fromeither clone, an obstacle attributed to constitutive levels ofexpression of DNase from the clones, so that these authors were unableto determine the sequence for the DNase-encoding nucleic acid. Accordingto Shields et al., the inability to recover the plasmid was the resultof constitutive expression of DNase even when the promoter was repressedat low temperature. This would create a considerable obstacle sinceShields et al. had only identified the clone by expression cloning,which necessarily requires that the DNase be placed under the control ofa promoter of some sort.

[0005] DNase finds a number of known utilities, and has been used fortherapeutic purposes. Its principal therapeutic use has been to reducethe viscosity of pulmonary secretions in such diseases as pneumonia,thereby aiding in the clearing of respiratory airways. Obstruction ofairways by secretions can cause respiratory distress, and in some cases,can lead to respiratory failure and death. Bovine pancreatic DNase hasbeen sold under the tradename Dornavac (Merck), but this product waswithdrawn from the market. Reports indicate that this product had someclinical efficacy. However, although some clinicians observed nosignificant side effects (Lieberman, JAMA 205:312 [1968]), others notedserious complications such as pulmonary irritation and anaphylaxis(Raskin, Am. Rev. Resp. Dis., 98:697 [1968]). Such complications may beattributed to the fact that the previously marketed products werecontaminated with proteases and were immunogenic in humans. In fact,although the clinical problem of thick pulmonary secretions is oftenchronic and recurring, prolonged therapy with bovine pancreatic DNasewas not recommended. These problems could be overcome by providing DNaseof human origin and producing it in large quantities in nonpancreaticexocrine cells to facilitate purification free of contaminant proteases.

[0006] Accordingly, it is an object of this invention to provide nucleicacid encoding human DNase.

[0007] It is another object to provide a method for expression of humanDNase in recombinant cell culture.

[0008] A further object is to enable the preparation of DNase havingvariant amino acid sequences or glycosylation not otherwise found innature, as well as other derivatives of DNase, having improvedproperties including enhanced specific activity.

SUMMARY OF THE INVENTION

[0009] The objects of this invention have been accomplished by a methodcomprising providing nucleic acid encoding human DNase; transforming ahost cell with the nucleic acid; culturing the host cell to allow DNaseto accumulate in the culture; and recovering DNase from the culture.Surprisingly, a full length clone encoding human DNase has beenidentified and recovered, and moreover this DNA is readily expressed byrecombinant host cells.

[0010] In preferred embodiments the mammalian DNase is full-length,mature human DNase, having the amino acid sequence of native humanDNase, its naturally occurring alleles, or predetermined amino acidsequence or glycosylation variants thereof. The nucleic acid encodingthe DNase preferably encodes a preprotein which is processed andsecreted from host cells, particularly mammalian cells.

BRIEF DESCRIPTION OF THE FIGURES

[0011]FIG. 1 depicts the amino acid and DNA sequence of human DNase. Thenative signal sequence is underlined, the potential initiation codonsare circled, and the mature sequence is bracketed.

[0012]FIG. 2 shows a comparison between the amino acid sequence formature human (hDNase) and bovine (bDNase) DNase. Asterisks denominateexact homology, periods designate conserved substitutions.

[0013]FIG. 3 shows the construction of the expression vectorspRK.DNase.3 and pSVe.DNase.

[0014]FIG. 4 shows the construction of the expression vector pSVI.DNasethat contains the splice unit of the pRK5 vector without anymodifications.

[0015]FIG. 5 shows the construction of the expression vectorspSVI12.DNase, pSVI3.DNase, pSVI5.DNase, and pSV16b.DNase containing themodifications in the splice unit and surrounding DNA.

[0016]FIG. 6 shows the complete nucleotide sequence of pSVI.DNase up to,but not including, the coding region of DNase.

[0017]FIG. 7 shows the complete nucleotide sequence of pSVI2.DNase upto, but not including, the coding region of DNase.

[0018]FIG. 8 shows the complete nucleotide sequence of pSVI3.DNase upto, but not including, the coding region of DNase.

[0019]FIG. 9 shows the complete nucleotide sequence of pSVI5.DNase upto, but not including, the coding region of DNase.

[0020]FIG. 10 shows the complete nucleotide sequence of pSVI6B.DNase upto, but not including, the coding region of DNase.

[0021]FIG. 11 shows a schematic representation of the splice unitnucleotide sequences involved in the preparation of the vectors of thisexample, i.e., SVI, SVI2, SVI3, SVI5, and SVI6B. The boxes representchanges from the SVI sequence, the double underlining is a spurious ATGcodon, the underlining shows spurious splice sites and added or changedbranchpoint sequence (BPS) regions, the breaks in sequence representdeletions of the nucleotides for SVI3-SVI5, the “ . . . ” designationindicates sequence not shown, and the carets indicate the 5′ and 3′cleavage sites within the splice donor and splice acceptor,respectively, of the splice unit.

DETAILED DESCRIPTION

[0022] Human DNase is defined as a polypeptide having the amino acidsequence of FIG. 1 together with amino acid sequence variants thereofwhich retain the qualitative enzymatic activity of DNase. Preferably,the variants are not immunogenic in humans. Variants may possess greaterenzymatic activity, enhanced resistance to inhibition (in particular byactin), improved solubility, or may be expressed at higher levels byhost cells.

[0023] Amino acid sequence variants of DNase fall into one or more ofthree classes: Substitutional, insertional or deletional variants. Thesevariants ordinarily are prepared by site specific mutagenesis ofnucleotides in the DNA encoding the DNase, by which DNA encoding thevariant is obtained, and thereafter expressing the DNA in recombinantcell culture. However, variant DNase fragments having up to about100-150 residues may be prepared conveniently by in vitro synthesis.

[0024] The amino acid sequence variants of human DNase are predeterminedor are naturally occurring alleles. For example, bovine pancreatic DNaseis found naturally as 4 molecular variants which possess the sameenzymatic activity but differ in glycosylation pattern or substitutionat the amino acid level. Additionally, human DNase is found naturallywith an arginine or a glutamine residue at amino acid 222. The variantstypically exhibit the same qualitative biological activity as thenaturally-occurring analogue. Specifically excluded from the scope ofhuman DNase as described herein are the sequences of naturally occurringbovine, porcine or ovine DNase.

[0025] While the site for introducing an amino acid sequence variationis predetermined, the mutation per se need not be predetermined. Forexample, in order to optimize the performance of a mutation at a givensite, saturation mutagenesis is introduced at the target codon or regionand the DNase variants then screened for the optimal combination ofdesired activity.

[0026] Amino acid substitutions are typically introduced for singleresidues; insertions usually will-be on the order of about from 1 to 10amino acid residues; and deletions will range about from 1 to 30residues. Deletions or insertions preferably are made in adjacent pairs,i.e. a deletion of 2 residues or insertion of 2 residues. Substitutions,deletions, insertions or any combination thereof may be combined toarrive at a final construct. Obviously, the mutations that will be madein the DNA encoding the variant DNase must not place the sequence out ofreading frame and preferably will not create complementary regions thatcould produce secondary mRNA structure (EP 75,444A).

[0027] Substitutional variants are those in which at least one residuein the FIG. 1 sequence has been removed and a different residue insertedin its place. Such substitutions generally are made in accordance withthe following Table 1 when it is desired to finely modulate thecharacteristics of DNase. TABLE 1 Original Residue ExemplarySubstitutions Ala ser Arg lys Asn gln; his Asp glu Cys ser Gln asn Gluasp Gly pro His asn; gln Ile leu; val Leu ile; val Lys arg; gln; glu Metleu; ile Phe met; leu; tyr Ser thr Thr ser Trp tyr Tyr trp; phe Val ile;ieu

[0028] Substantial changes in function or immunological identity aremade by selecting substitutions that are less conservative than those inTable 1, i.e., selecting residues that differ more significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site or (c) the bulk of the side chain. The substitutionswhich in general are expected to produce the greatest changes in DNaseproperties will be those in which (a) a hydrophilic residue, e.g. serylor threonyl, is substituted for (or by) a hydrophobic residue, e.g.leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine orproline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain, e.g., lysyl, arginyl, or histidyl,is substituted for (or by) an electronegative residue, e.g., glutamyl oraspartyl; or (d) a residue having a bulky side chain, e.g.,phenylalanine, is substituted for (or by) one not having a side chain,e.g., glycine.

[0029] Examples of human DNase amino acid sequence variants aredescribed in the table below. The residue following the residue numberindicates the replacement or inserted amino acids. TABLE 2 SubstitutionsH134K, N, R or Q FGW, L, I, M or V A135S I8F, L, V, M or W L133V, I or AR41K or H P132A S43T, C or Y N18K, H, R or Q K77R or H S17T R79K or HT20S or Y N110D, S, T, R, Y or Q N106K, H, R or Q G167P G105P or AF169L, V, I or M D107E or T A171G, V or M R140A S250T, Y or M R12lK or HY253T, S or M T108S or Y R73N P103S, T or Y R73C + D139C C101S, T, Y orM K260R or S C104S, T, Y or M L259V, I or M Deletions InsertionsΔ34H-Δ39E K260RA-COOH Δ159G-161E V131A P132 Δ204A-208H P132A L133Δ12lR-127E Δ188T-191T Δ204A-208H Δ223G-231L Δ260K

[0030] In general, sequence variation is introduced into residues 6-10,41-43, 77-79, 110-112, 167-171, 250-253, 73, 93, 157, 149, 185, 187,198, 17-20, 105-108 and 131-139. Preferably, the variants representconservative substitutions. It will be understood that some variants mayexhibit reduced or absent hydrolytic activity. These variantsnonetheless are useful as standards in immunoassays for DNase so long asthey retain at least one immune epitope of native human DNase.

[0031] Glycosylation variants are included within the scope of humanDNase. Included are unglycosylated amino acid sequence variants,unglycosylated DNase having the native, unmodified amino acid sequenceof DNase, and glycosylation variants. For example, substitutional ordeletional mutagenesis is employed to eliminate the N-linkedglycosylation sites of human DNase found at residues 18 and 106, e.g.,the asparagine residue is deleted or substituted for by another basicresidue such as lysine or histidine. Alternatively, flanking residuesmaking up the glycosylation site are substituted or deleted, even thoughthe asparagine residues remain unchanged, in order to preventglycosylation by eliminating the glycosylation recognition site.Unglycosylated DNase which has the amino acid sequence of native humanDNase is produced in recombinant prokaryotic cell culture becauseprokaryotes are incapable of introducing glycosylation intopolypeptides. In addition, glycosylation variants may be generated byadding potential N-linked glycosylation sites through inserting (eitherby amino acid substitution or deletion) consensus sequences for N-linkedglycosylation: Asn-X-Ser or Asn-X-Thr. Glycosylation variants may begenerated by both eliminating the N-linked glycosylation sites atresidues 18 and 106 and by adding new ones.

[0032] Glycosylation variants, i.e., glycosylation which is differentfrom that of human pancreatic or urinary DNase, are produced byselecting appropriate host cells or by in vitro methods. Yeast, forexample, introduce glycosylation which varies significantly from that ofmammalian systems. Similarly, mammalian cells having a different species(e.g. hamster, murine, insect, porcine, bovine or ovine) or tissueorigin (e.g. lung, liver, lymphoid, mesenchymal or epidermal) than thesource of the DNase are screened for the ability to introduce variantglycosylation as characterized for example by elevated levels of mannoseor variant ratios of mannose, fucose, sialic acid, and other sugarstypically found in mammalian glycoproteins. In addition, mammalian oryeast cells which possess mutations with respect to glycosylationphenotype may be identified, selected for following mutation, orconstructed and utilized to produce DNase. In vitro processing of DNasetypically is accomplished by enzymatic hydrolysis, e.g. neuramimidase orendoglycosydase H digestion.

[0033] Insertional amino acid sequence variants of DNases are those inwhich one or more amino acid residues are introduced into apredetermined site in the target DNase and which displace thepreexisting residues. Most commonly, insertional variants are fusions ofheterologous proteins or polypeptides to the amino or carboxyl terminusof DNase. DNase derivatives which are immunogenic in humans are notpreferred, e.g. those which are made by fusing an immunogenicpolypeptide to DNase by cross-linking in vitro or by recombinant cellculture transformed with DNA encoding immunogenic fusions such as lacZ.For this reason, the typical insertional variants contemplated hereinare the signal sequence variants described above.

[0034] Covalent modifications of the DNase molecule are included withinthe scope hereof. Such modifications are introduced by reacting targetedamino acid residues of the recovered protein with an organicderivatizing agent that is capable of reacting with selected side chainsor terminal residues, or by harnessing mechanisms of post-translationalmodification that function in selected recombinant host cells. Theresulting covalent derivatives are useful in programs directed atidentifying residues important for biological activity, for immunoassaysof DNase or for the preparation of anti-human DNase antibodies forimmunoaffinity purification of recombinant DNase. For example, completeinactivation of the biological activity of the protein after reactionwith ninhydrin would suggest that at least one arginyl or lysyl residueis critical for its activity, whereafter the individual residues whichwere modified under the conditions selected are identified by isolationof a peptide fragment containing the modified amino acid residue.

[0035] Cysteinyl residues most commonly are reacted with α-haloacetates(and corresponding amines), such as chloracetic acid or chloroacetamideto give carboxymethyl or carboxamidomethyl derivatives. Cysteinylresidues also are derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl) propionic acid, chloroacetol phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol orchloro-nitrobenzo-2-oxa-1,3-diazole.

[0036] Histidyl residues preferably are derivatized by reaction withdiethylpyrocarbonate at pH 5.5 to 7.0 because this agent is relativelyspecific for the histidyl side chain. Para-bromo-phenacyl bromide alsois useful; the reaction should be performed in 0.1M sodium cacodylate atpH 6.0.

[0037] Lysinyl and amino terminal residues are reacted with succinic orother carboxylic acid anhydrides. Derivatization with these agents hasthe effect of reversing the charge of the lysinyl residues. Othersuitable reagents for derivatizing a amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; cloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

[0038] Arginyl residues are modified by reaction with one of severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pKa of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineε-amino group.

[0039] The specific modification of tyrosyl residues per se has beenextensively studied, with particular interest in introducing spectrallabels into tyrosyl residues by reaction with aromatic diazoniumcompounds or tetranitromethane. Most commonly, N-acetylimidizol andtetranitromethane are used to form O-acetyl tyrosyl species and 3-nitroderivatives, respectively. Tyrosyl residues are iodinated using ¹²⁵I or¹³¹I to prepare labelled proteins for use in radioimmunoassay, thechloramine T method being widely adopted per se for this purpose.

[0040] Carboxyl side groups (aspartyl or glutamyl) are selectivelymodified by reaction with carbodiimides (R′—N═C═N—R′) such as1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl)-carbodiimide. Furthermore,aspartyl and glutamyl residues are converted to asparaginyl andglutaminyl residues by reaction with ammonium ions, this being analternative to mutating the nucleic acid to encode asparagine areglutamine.

[0041] Derivatization with bifunctional agents is useful for preparingintermolecular aggregates of the protein with immunogenic polypeptidesas well as for cross-linking the protein to a water insoluble supportmatrix or surface for use in the assay or affinity purification ofantibody. In addition, a study of intrachain cross-links will providedirect information on conformational structure. Commonly usedcross-linking agents include 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, for example esters with4-azidosalicylic acid, homobifunctional imidoesters includingdisuccinimidyl esters such as 3,3′-dithiobis (succinimidyl-propionate),and bifunctional maleimides such as bis-N-maleimido-1,8-octane.Derivatizing agents such as methyl-3-[(p-azido-phenyl)dithio]propioimidate yield photoactivatable intermediates which are capable offorming cross-links an the presence of light. Alternatively, reactivewater insoluble matrices such as cyanogen bromide activatedcarbohydrates and the reactive substrates described in U.S. Pat. Nos.3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537 and 4,330,440 areemployed for protein immobilization.

[0042] Certain post-translational derivatizations are the result of theaction of recombinant host cells on the expressed polypeptide.Glutaminyl and asparaginyl residues are frequently post-translationallydeamidated to the corresponding glutamyl and aspartyl residues.Alternatively, these residues are deamidated under mildly acidicconditions. Either form of these residues falls within the scope of thisinvention.

[0043] Other post-translational modifications include hydroxylation ofproline and lysine, phosphorylation of hydroxyl groups of seryl orthreonyl residues, methylation of the α-amino groups of lysine,arginine, and hystidine side chains (T. E. Creighton, Proteins:Structure and Molecular Properties, W. H. Freeman & Co., San Franciscopp 79-86 [1983]), acetylation of the N-terminal amine and, in someinstances, amidation of the C-terminal carboxyl.

[0044] Other derivatives comprise the polypeptide of this inventioncovalently bonded to a nonproteinaceous polymer. The nonproteinaceouspolymer ordinarily is a hydrophilic synthetic polymer, i.e., a polymernot otherwise found in nature. However, polymers which exist in natureand are produced by recombinant or in vitro methods are useful, as arepolymers which are isolated from nature. Hydrophilic polyvinyl polymersfall within the scope of this invention, e.g. polyvinylalcohol andpolyvinylpyrrolidone. Particularly useful are polyalkylene ethers suchas polyethylene glycol, polypropylene glycol, polyoxyethylene esters ormethoxy polyethylene glycol; polyoxyalkylenes such as polyoxyethylene,polyoxypropylene, and block copolymers of polyoxyethylene andpolyoxypropylene (Pluronics); polymethacrylates; carbomers; branched orunbranched polysaccharides which comprise the saccharide monomersD-mannose, D- and L-galactose, fucose, fructose, D-xylose, L-arabinose,D-glucuronic acid, sialic acid, D-galacturonic acid, D-mannuronic acid(e.g. polymannuronic acid, or alginic acid), D-glucosamine,D-galactosamine, D-glucose and neuraminic acid includinghomopolysaccharides and heteropolysaccharides such as lactose,amylopectin, starch, hydroxyethyl starch, amylose, dextran sulfate,dextran, dextrins, glycogen, or the polysaccharide subunit of acidmucopolysaccharides, e.g. hyaluronic acid; polymers of sugar alcoholssuch as polysorbitol and polymannitol; and heparin or heparon. Where thepolysaccharide is the native glycosylation or the glycosylationattendant on recombinant expression, the site of substitution may belocated at other than a native N or O-linked glycosylation site whereinan additional or substitute N or O-linked site has been introduced intothe molecule. Mixtures of such polymers are employed, or the polymer maybe homogeneous. The polymer prior to crosslinking need not be, butpreferably is, water soluble, but the final conjugate must be watersoluble. In addition, the polymer should not be highly immunogenic inthe conjugate form, nor should it possess viscosity that is incompatiblewith intravenous infusion or injection if it is intended to beadministered by such routes.

[0045] Preferably the polymer contains only a single group which isreactive. This helps to avoid cross-linking of protein molecules.However, it is within the scope herein to optimize reaction conditionsto reduce cross-linking, or to purify the reaction products through gelfiltration or chromatographic sieves to recover substantiallyhomogeneous derivatives.

[0046] The molecular weight of the polymer ranges about from 100 to500,000, and preferably is about from 1,000 to 20,000. The molecularweight chosen will depend upon the nature of the polymer and the degreeof substitution. In general, the greater the hydrophilicity of thepolymer and the greater the degree of substitution, the lower themolecular weight that can be employed. Optimal molecular weights will bedetermined by routine experimentation.

[0047] The polymer generally is covalently linked to the polypeptideherein through a multifunctional crosslinking agent which reacts withthe polymer and one or more amino acid or sugar residues of the protein.However, it is within the scope of this invention to directly crosslinkthe polymer by reacting a derivatized polymer with the protein, or viceversa.

[0048] The covalent crosslinking site on the polypeptide includes theN-terminal amino group and epsilon amino groups found on lysineresidues, as well as other amino, imino, carboxyl, sulfhydryl, hydroxylor other hydrophilic groups. The polymer may be covalently bondeddirectly to the protein without the use of a multifunctional (ordinarilybifunctional) crosslinking agent. Examples of such crosslinking agentsinclude 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example esters with 4-azidosalicylicacid, homobifunctional imidoesters including disuccinimidyl esters suchas 3,3′-dithiobis (succinimidyl-propionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane. Derivatizing agents such asmethyl-3-[(p-azido-phenyl)dithio] propioimidate yield photoactivatableintermediates which are capable of forming cross-links in the presenceof light. Alternatively, reactive water soluble matrices such ascyanogen bromide activated carbohydrates and the systems described inU.S. Pat. Nos. 3,959,080; 3,969,287; 3,691,016; 4,195,128; 4,247,642;4,229,537; 4,055,635 and 4,330,440 are suitably modified forcross-linking. Covalent bonding to amino groups is accomplished by knownchemistries based upon cyanuric chloride, carbonyl diimidazole, aldehydereactive groups (PEG alkoxide plus diethyl acetal of bromoacetaldehyde;PEG plus DMSO and acetic anhydride, or PEG chloride plus the phenoxideof 4-hydroxybenzaldehyde, succinimidyl active esters, activateddithiocarbonate PEG, 2,4,5-trichlorophenylchloroformate orp-nitrophenylchloroformate activated PEG. Carboxyl groups arederivatized by coupling PEG-amine using carbodiimide. Polymers areconjugated to oligosaccharide groups by oxidation using chemicals, e.g.metaperiodate, or enzymes, e.g. glucose or galactose oxidase, (either ofwhich produces the aldehyde derivative of the carbohydrate), followed byreaction with hydrazide or amino-derivatized polymers, in the samefashion as is described by Heitzmann et al., P.N.A.S., 71:3537-3541(1974) or Bayer et al., Methods in Enzymology, 62:310 (1979), for thelabeling of oligosaccharides with biotin or avidin. Further, otherchemical or enzymatic methods which have been used heretofore to linkoligosaccharides and polymers may be suitable. Substitutedoligosaccharides are particularly advantageous because, in general,there are fewer substitutions than amino acid sites for derivatization,and the oligosaccharide products thus will be more homogeneous. Theoligosaccharide substituents also are optionally modified by enzymedigestion to remove sugars, e.g. by neuramimidase digestion, prior topolymer derivatization.

[0049] The polymer will bear a group which is directly reactive with anamino acid side chain, or the N- or C-terminus of the polypeptideherein, or which is reactive with the multifunctional cross-linkingagent. In general, polymers bearing such reactive groups are known forthe preparation of immobilized proteins. In order to use suchchemistries here, one should employ a water soluble polymer otherwisederivatized in the same fashion as insoluble polymers heretoforeemployed for protein immobilization. Cyanogen bromide activation is aparticularly useful procedure to employ in crosslinking polysaccharides.

[0050] “Water soluble” in reference to the polymer conjugate means thatthe conjugate is soluble in physiological fluids such as blood in anamount which is sufficient to achieve a therapeutically effectiveconcentration.

[0051] “Water soluble” in reference to the starting polymer means thatthe polymer or its reactive intermediate used for conjugation issufficiently water soluble to participate in a derivatization reaction.

[0052] The degree of substitution with polymer will vary depending uponthe number of reactive sites on the protein, whether all or a fragmentof protein is used, whether the protein is a fusion with a heterologousprotein, the molecular weight, hydrophilicity and other characteristicsof the polymer, and the particular protein derivatization sites chosen.In general, the conjugate contains about from 1 to 10 polymer molecules,while any heterologous sequence may be substituted with an essentiallyunlimited number of polymer molecules so long as the desired activity isnot significantly adversely affected. The optimal degree of crosslinkingis easily determined by an experimental matrix in which the time,temperature and other reaction conditions are varied to change thedegree of substitution, after which the ability of the conjugates tofunction in the desired fashion is determined.

[0053] The polymer, e.g. PEG, is crosslinked by a wide variety ofmethods known per se for the covalent modification of proteins withnonproteinaceous polymers such as PEG. Certain of these methods,however, are not preferred for the purposes herein. Cyanuric chloridechemistry leads to many side reactions, including protein cross-linking.In addition, it may be particularly likely to lead to inactivation ofproteins containing sulfhydryl groups. Carbonyl diimidazole chemistry(Beauchamp et al., “Anal. Biochem.” 131:25-33 [1983]) requires high pH(>8.5), which can inactivate proteins. Moreover, since the “activatedPEG” intermediate can react with water, a very large molar excess of“activated PEG” over protein is required. The high concentrations of PEGrequired for the carbonyl diimidazole chemistry also led to problemswith purification, as both gel filtration chromatography and hydrophobicinteraction chromatography are adversely effected. In addition, the highconcentrations of “activated PEG” may precipitate protein, a problemthat per se has been noted previously (Davis, U.S. Pat. No. 4,179,337).On the other hand, aldehyde chemistry (Royer, U.S. Pat. No. 4,002,531)is more efficient since it requires only a 40 fold molar excess of PEGand a 1-2 hr incubation. However, the manganese dioxide suggested byRoyer for preparation of the PEG aldehyde is problematic “because of thepronounced tendency of PEG to form complexes with metal-based oxidizingagents” (Harris et al., “J. Polym. Sci., Polym. Chem. Ed.” 22:341-352[1984]). The use of a moffatt oxidation, utilizing DMSO and aceticanhydride, obviates this problem. In addition, the sodium borohydridesuggested by Royer must be used at a high pH and has a significanttendency to reduce disulfide bonds. In contrast, sodiumcyanoborohydride, which is effective at neutral pH and has very littletendency to reduce disulfide bonds is preferred.

[0054] The conjugates of this invention are separated from unreactedstarting materials by gel filtration. Heterologous species of theconjugates are purified from one another in the same fashion.

[0055] The polymer also may be water insoluble, as a hydrophilic gel ora shaped article. Particularly useful are polymers comprised by surgicaltubing such as catheters or drainage conduits.

[0056] DNA encoding human DNase is synthesized by in vitro methods or isobtained readily from human pancreatic cDNA libraries. Since FIG. 1gives the entire DNA sequence for human DNase, one needs only to conducthybridization screening with labelled DNA encoding human DNase orfragments thereof (usually, greater than about 50 bp) in order to detectclones in the cDNA libraries which contain homologous sequences,followed by analyzing the clones by restriction enzyme analysis andnucleic acid sequencing to identify full-length clones. If full lengthclones are not present in the library, then appropriate fragments may berecovered from the various clones and ligated at restriction sitescommon to the fragments to assemble a full-length clone. DNA encodingDNase from other animal species is obtained by probing libraries fromsuch species with the human sequence, or by synthesizing the genes invitro (for bovine, porcine or ovine DNase).

[0057] Included within the scope hereof are nucleic acid probes whichare capable of hybridizing under high stringency conditions to the cDNAof human DNase or to the genomic gene for human DNase (including intronsand 5′ or 3′ flanking regions extending to the adjacent genes or about5,000 bp, whichever is greater). Identification of the genomic DNA forDNase is a straight-forward matter of probing a human genomic librarywith the cDNA or its fragments which have been labelled with adetectable group, e.g. radiophosphorus, and recovering clone(s)containing the gene. The complete gene is pieced together by “walking”if necessary. Typically, the probes do not encode bovine, ovine orporcine DNase, and they range about from 10 to 100 bp in length.

[0058] In general, prokaryotes are used for cloning of DNA sequences inconstructing the vectors useful in the invention. For example, E. coliK12 strain 294 (ATCC No. 31446) is particularly useful. Other microbialstrains which may be used include E. coli B and E. coli X1776 (ATCC No.31537). These examples are illustrative rather than limiting.Alternatively, in vitro methods of cloning, e.g., PCR, are suitable.

[0059] DNase is expressed directly in recombinant cell culture as anN-terminal methionyl analogue, or as a fusion with a polypeptideheterologous to human DNase, preferably a signal sequence or otherpolypeptide having a specific cleavage site at the N-terminus of theDNase. For example, in constructing a prokaryotic secretory expressionvector the native DNase signal is employed with hosts that recognize thehuman signal. When the secretory leader is “recognized” by the host, thehost signal peptidase is capable of cleaving a fusion of the leaderpolypeptide fused at its C-terminus to the desired mature DNase. Forhost prokaryotes that do not process the human signal, the signal issubstituted by a prokaryotic signal selected for example from the groupof the alkaline phosphatase, penicillinase, lpp or heat stableenterotoxin II leaders. For yeast secretion the human DNase signal maybe substituted by the yeast invertase, alpha factor or acid phosphataseleaders. In mammalian cell expression the native signal is satisfactory,although other mammalian secretory protein signals are suitable, as areviral secretory leaders, for example the herpes simplex gD signal.

[0060] DNase is expressed in any host cell, but preferably issynthesized in mammalian hosts. However, host cells from prokaryotes,fungi, yeast, pichia, insects and the like are also are used forexpression. Exemplary prokaryotes are the strains suitable for cloningas well as E. coli W3110 (F⁻, λ⁻ prototrophic, ATTC No. 27325), otherenterobacteriaceae such as Serratia marcescans, bacilli and variouspseudomonads. Preferably the host cell should secrete minimal amounts ofproteolytic enzymes.

[0061] Expression hosts typically are transformed with DNA encodinghuman DNase which has been ligated into an expression vector. Suchvectors ordinarily carry a replication site (although this is notnecessary where chromosomal integration will occur). It is presentlypreferred to utilize an expression vector as described in Example 4below, where the vector contains a splice-donor-intron-splice-acceptorsequence or unit.

[0062] Expression vectors also include marker sequences which arecapable of providing phenotypic selection in transformed cells. Forexample, E. coli is typically transformed using pBR322, a plasmidderived from an E. coli species (Bolivar, et al., Gene 2: 95 [1977]).pBR322 contains genes for ampicillin and tetracycline resistance andthus provides easy means for identifying transformed cells, whether forpurposes of cloning or expression. Expression vectors also optimallywill contain sequences which are useful for the control of transcriptionand translation, e.g., promoters and Shine-Dalgarno sequences (forprokaryotes) or promoters and enhancers (for mammalian cells). Thepromoters may be, but need not be, inducible; surprisingly, evenpowerful constitutive promoters such as the CMV promoter for mammalianhosts have been found to produce DNase without host cell toxicity. Whileit is conceivable that expression vectors need not contain anyexpression control, replicative sequences or selection genes, theirabsence may hamper the identification of DNase transformants and theachievement of high level DNase expression.

[0063] Promoters suitable for use with prokaryotic hosts illustrativelyinclude the β-lactamase and lactose promoter systems (Chang et al.,“Nature”, 275: 615 [1978]; and Goeddel et al., “Nature” 281: 544[1979]), alkaline phosphatase, the tryptophan (trp) promoter system(Goeddel “Nucleic Acids Res.” 8: 4057 [1980] and EPO Appln. Publ. No.36,776) and hybrid promoters such as the tac promoter (H. de Boer etal., “Proc. Natl. Acad. Sci. USA” 80: 21-25 [1983]). However, otherfunctional bacterial promoters are suitable. Their nucleotide sequencesare generally known, thereby enabling a skilled worker operably toligate them to DNA encoding DNase (Siebenlist et al., “Cell” 20: 269[1980]) using linkers or adaptors to supply any required restrictionsites. Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encodingDNase.

[0064] In addition to prokaryotes, eukaryotic microbes such as yeast, orfilamentous fungi are satisfactory. Saccharomyces cerevisiae is the mostcommonly used eukaryotic microorganism, although a number of otherstrains are commonly available. Strains of Saccharomyces cerevisiaehaving the identifying characteristics of strain AB107-30(4)-VN#2 (ATCCAccession No. 20937), particularly its resistance to 4M orthovanadate,is particularly suitable for DNase expression (see U.S. Ser. No.07/343,863, filed Apr. 26, 1989, specifically incorporated byreference). With this VN#2 strain, it is desirable to stably transformthe cells with a high-copy-number plasmid derived from a yeast 2-micronplasmid, id. Generally, the plasmid YRp7 is a satisfactory expressionvector in yeast (Stinchcomb, et al., Nature 282: 39 [1979]; Kingsman etal., Gene 7: 141 [1979]; Tschemper et al. , Gene 10: 157 [1980]). Thisplasmid already contains the trp1 gene which provides a selection markerfor a mutant strain of yeast lacking the ability to grow in tryptophan,for example ATCC no. 44076 or PEP4-1 (Jones, Genetics 85: 12 [1977]).The presence of the trp1 lesion as a characteristic of the yeast hostcell genome then provides an effective environment for detectingtransformation by growth in the absence of tryptophan. Expression inpichia (U.S. Pat. No. 4,808,537) is also satisfactory.

[0065] Suitable promoting sequences for use with yeast hosts include thepromoters for 3-phosphoglycerate kinase (Hitzeman et al., “J. Biol.Chem.” 255: 2073 [1980]) or other glycolytic enzymes (Hess et al., “J.Adv. Enzyme Reg.” 7: 149 [1968]; and Holland, “Biochemistry” 17: 4900[1978]), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

[0066] Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin R. Hitzeman et al., European Patent Publication No. 73,657A.

[0067] Expression control sequences are known for eukaryotes. Virtuallyall eukaryotic genes have an AT-rich region located approximately 25 to30 bases upstream from the site where transcription is initiated.Another sequence found 70 to 80 bases upstream from the start oftranscription of many genes is a CXCAAT region where X may be anynucleotide. At the 3′ end of most eukaryotic genes is an AATAAA sequencewhich may be the signal for addition of the poly A tail to the 3′ end ofthe coding sequence. All of these sequences may be inserted intomammalian expression vectors.

[0068] Suitable promoters for controlling transcription from vectors inmammalian host cells are readily obtained from various sources, forexample, the genomes of viruses such as polyoma virus, SV40, adenovirus,MMV (steroid inducible), retroviruses (e.g. the LTR of HIV), hepatitis-Bvirus and most preferably cytomegalovirus, or from heterologousmammalian promoters, e.g. the beta actin promoter. The early and latepromoters of SV40 are conveniently obtained as an SV40 restrictionfragment which also contains the SV40 viral origin of replication. Fierset al., Nature, 273: 113 (1978). The immediate early promoter of thehuman cytomegalovirus is conveniently obtained as a HindIII Erestriction fragment. Greenaway, P. J. et al., Gene 18: 355-360 (1982).

[0069] Transcription of a DNA encoding DNase by higher eukaryotes isincreased by inserting an enhancer sequence into the vector. Enhancersare cis-acting elements of DNA, usually about from 10-300 bp, that acton a promoter to increase its transcription. Enhancers are relativelyorientation and position independent having been found 5′ (Laimins, L.et al., PNAS 78: 993 [1981]) and 3′ (Lusky, M. L., et al., Mol. CellBio. 3: 1108 [1983]) to the transcription unit, within an intron(Banerji, J. L. et al., Cell 33: 729 [1983]) as well as within thecoding sequence itself (Osborne, T. F., et al., Mol. Cell Bio. 4: 1293[1984]). Many enhancer sequences are now known from mammalian genes(globin, elastase, albumin, α-fetoprotein and insulin). Typically,however, one will use an enhancer from a eukaryotic cell virus. Examplesinclude the SV40 enhancer on the late side of the replication origin (bp100-270), the cytomegalovirus early promoter enhancer, the polyomaenhancer on the late side of the replication origin, and adenovirusenhancers.

[0070] Expression vectors used in eukaryotic host cells (yeast, fungi,insect, plant, animal, human or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription which may affect mRNA expression. These regions aretranscribed as polyadenylated segments in the untranslated portion ofthe mRNA encoding DNase. The 3′ untranslated regions also includetranscription termination sites.

[0071] Expression vectors may contain a selection gene, also termed aselectable marker. Examples of suitable selectable markers for mammaliancells are dihydrofolate reductase (DHFR), thymidine kinase (TK) orneomycin. When such selectable markers are successfully transferred intoa mammalian host cell, the transformed mammalian host cell is able tosurvive if placed under selective pressure. There are two widely useddistinct categories of selective regimes. The first category is based ona cell's metabolism and the use of a mutant cell line which lacks theability to grow independent of a supplemented media. Two examples are:CHO DHFR⁻ cells and mouse LTK⁻ cells. These cells lack the ability togrow without the addition of such nutrients as thymidine orhypoxanthine. Because these cells lack certain genes necessary for acomplete nucleotide synthesis pathway, they cannot survive unless themissing nucleotides are provided in a supplemented media. An alternativeto supplementing the media is to introduce an intact DHFR or TK geneinto cells lacking the respective genes, thus altering their growthrequirements. Individual cells which were not transformed with the DHFRor TK gene will not be capable of survival in non supplemented media.

[0072] The second category is dominant selection which refers to aselection scheme used in any cell type and does not require the use of amutant cell line. These schemes typically use a drug to arrest growth ofa host cell. Those cells which are successfully transformed with aheterologous gene express a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin (Southern et al. , J. Molec. Appl. Genet. 1: 327(1982)), mycophenolic acid (Mulligan et al. , Science 209: 1422 (1980))or hygromycin (Sugden et al., Mol. Cell. Biol. 5: 410-413 (1985)). Thethree examples given above employ bacterial genes under eukaryoticcontrol to convey resistance to the appropriate drug G418 or neomycin(geneticin), xgpt (mycophenolic acid) or hygromycin, respectively.

[0073] Suitable eukaryotic host cells for expressing human DNase includemonkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham, F. L. et al ., J. Gen Virol. 36: 59 [1977]);baby hamster kidney cells (BHK, ATCC CCL 10); chinese hamsterovary-cells-DHFR (CHO, Urlaub and Chasin, PNAS (USA) 77: 4216, [1980]);mouse sertoli cells (TM4, Mather, J. P., Biol. Reprod. 23: 243-251[1980]); monkey kidney cells (CV1 ATCC CCL 70); african green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); and, TR1 cells (Mather, J. P. et al., Annals N.Y.Acad. Sci. 383: 44-68 [1982]).

[0074] Construction of suitable vectors containing the desired codingand control sequences employ standard ligation techniques. Isolatedplasmids or DNA fragments are cleaved, tailored, and religated in theform desired to form the plasmids required.

[0075] For analysis to confirm correct sequences in plasmidsconstructed, the ligation mixtures are used to transform E. coli K12strain 294 (ATCC 31446) and successful transformants selected byampicillin or tetracycline resistance where appropriate. Plasmids fromthe transformants are prepared, analyzed by restriction and/or sequencedby the method of Messing et al., Nucleic Acids Res. 9: 309 (1981) or bythe method of Maxam et al., Methods in Enzymology 65: 499 (1980).

[0076] Host cells are transformed with the expression vectors of thisinvention and cultured in conventional nutrient media modified asappropriate for inducing promoters, selecting transformants oramplifying the DNase gene. The culture conditions, such as temperature,pH and the like, are those previously used with the host cell selectedfor expression, and will be apparent to the ordinarily skilled artisan.

[0077] “Transformation” means introducing DNA into an organism so thatthe DNA is replicable, either as an extrachromosomal element or bychromosomal integration. Unless indicated otherwise, the method usedherein for transformation of the host cells is the method of Graham, F.and van der Eb, A., Virology 52: 456-457 (1973). However, other methodsfor introducing DNA into cells such as by nuclear injection or byprotoplast fusion may also be used. If prokaryotic cells or cells whichcontain substantial cell wall constructions are used, the preferredmethod of transfection is calcium treatment using calcium chloride asdescribed by Cohen, F. N. et al., Proc. Natl. Acad. Sci. (USA), 69: 2110(1972).

[0078] “Transfection” refers to the introduction of DNA into a host cellwhether or not any coding sequences are ultimately expressed. Numerousmethods of transfection are known to the ordinarily skilled artisan, forexample, CaPO₄ and electroporation. Transformation of the host cell isthe indicia of successful transfection.

[0079] DNase is recovered and purified from recombinant cell cultures bymethods used heretofore with human, bovine, ovine, or porcine DNase,including ammonium sulfate or ethanol precipitation, acid extraction,anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography (e.g. using DNA or nucleotides on a solid support),hydroxyapatite chromatography and lectin chromatography. Moreover,reverse-phase HPLC and chromatography using anti-DNase antibodies areuseful for the purification of DNase. As noted previously, (Price et al., J. Biol. Chem. 244:917 [1969]) it is preferred to have lowconcentrations (approximately 0.1-5 mM) of calcium ion present duringpurification. Other divalent cations which stabilize DNase are alsoutilized. DNase may be purified in the presence of a protease inhibitorsuch as PMSF.

[0080] Human DNase is placed into therapeutic formulations together withrequired cofactors, and optionally is administered in the same fashionas has been the case for animal DNase such as bovine pancreatic DNase.The formulation of DNase may be liquid, and is preferably an isotonicsalt solution such as 150 mM sodium chloride, containing 1.0 mM calciumat pH 7. The concentration of sodium chloride may range from 75-250 mM.The concentration of calcium may range from 0.01-5 mM, and otherdivalent cations which stabilize DNase may be included or substitutedfor calcium. The pH may range from 5.5-9.0, and buffers compatible withthe included divalent cation may also be utilized. The formulation maybe lyophilized powder, also containing calcium.

[0081] Commercially available nebulizers for liquid formulations,including jet nebulizers and ultrasonic nebulizers are useful foradministration. Liquid formulations may be directly nebulized andlyophilized power nebulized after reconstitution. Alternatively, DNasemay be aerosolized using a fluorocarbon formulation and a metered doseinhaler, or inhaled as a lyophilized and milled powder. In addition, theliquid formulation of DNase may be directly instilled in thenasotracheal or endotracheal tubes in intubated patients.

[0082] The purified DNase is employed for enzymatic alteration of thevisco-elasticity or the stickiness of mucous. Human DNase isparticularly useful for the treatment of patients with pulmonary diseasewho have abnormal, viscous or inspissated purulent secretions inconditions such as acute or chronic bronchopulmonary disease (infectiouspneumonia, bronchitis or tracheobronchitis, bronchiectasis, cysticfibrosis, asthma, TB or fungal infections), atelectasis due to trachealor bronchial impaction, and complications of tracheostomy. For suchtherapies a solution or finely divided dry preparation of human DNase isinstilled in conventional fashion into the bronchi, e.g. byaerosolization of a solution of DNase. Human DNase is also useful foradjunctive treatment for improved management of abscesses or severeclosed-space infections in conditions such as empyema, meningitis,abscess, peritonitis, sinusitis, otitis, periodontitis, pericarditis,pancreatitis, cholelithiasis, endocarditis and septic arthritis, as wellas in topical treatments in a variety of inflammatory and infectedlesions such as infected lesions of the skin and/or mucosal membranes,surgical wounds, ulcerative lesions and burns. Human DNase finds utilityin maintaining the flow in medical conduits communicating with a bodycavity, including surgical drainage tubes, urinary catheters, peritonealdialysis ports, and intratracheal oxygen catheters. DNase may improvethe efficacy of antibiotics in infections (e.g., gentamicin activity ismarkedly reduced by reversible binding to intact DNA). It also may beuseful as an oral supplement in cases of pancreatic insufficiency. DNasewill be useful in degrading DNA contaminants in pharmaceuticalpreparations: the preparation is contacted with DNase under conditionsfor degrading the contaminant DNA to oligonucleotide and thereafterremoving the oligonucleotide and DNase from the preparation. Use ofDNase immobilized on a water insoluble support is convenient in thisutility. Finally, DNase may be useful in treating non-infectedconditions in which there is an accumulation of cellular debris,including cellular DNA. For example, DNase would be useful aftersystemic administration in the treatment of pyelonephritis andtubulo-interstitial kidney disease (e.g., with blocked tubules secondaryto cellular debris), including drug-induced nephropathy or acute tubularnecrosis.

[0083] DNase may also be administered along with other pharmacologicagents used to treat the conditions listed above, such as antibiotics,bronchodilators, anti-inflammatory agents, and mucolytics (e.g.n-acetyl-cysteine). It may also be useful to administer DNase along withother therapeutic human proteins such as growth hormone, proteaseinhibitors, gamma-interferon, enkephalinase, lung surfactant, and colonystimulating factors.

[0084] In order to facilitate understanding of the following examplescertain frequently occurring methods and/or terms will be described.

[0085] “Plasmids” are designated by a lower case p preceded and/orfollowed by capital letters and/or numbers. The starting plasmids hereinare either commercially available, publicly available on an unrestrictedbasis, or can be constructed from available plasmids in accord withpublished procedures. In addition, equivalent plasmids to thosedescribed are known in the art and will be apparent to the ordinarilyskilled artisan.

[0086] “Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

[0087] Size separation of the cleaved fragments is performed using 8percent polyacrylamide gel described by Goeddel, D. et al., NucleicAcids Res., 8: 4057 (1980).

[0088] “Dephosphorylation” refers to the removal of the terminal 5′phosphates by treatment with bacterial alkaline phosphatase (BAP). Thisprocedure prevents the two restriction cleaved ends of a DNA fragmentfrom “circularizing” or forming a closed loop that would impedeinsertion of another DNA fragment at the restriction site. Proceduresand reagents for dephosphorylation are conventional. Maniatis, T. et al., Molecular Cloning pp. 133-134 (1982). Reactions using BAP are carriedout in 50 mM Tris at 68° C. to suppress the activity of any exonucleaseswhich may be present in the enzyme preparations. Reactions were run for1 hour. Following the reaction the DNA fragment is gel purified.

[0089] “Oligonucleotides” refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5′ phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

[0090] “Ligation” refers to the process of forming phosphodiester bondsbetween two double stranded nucleic acid fragments (Maniatis, T. et al.,Id., p. 146). Unless otherwise provided, ligation may be accomplishedusing known buffers and conditions with 10 units of T4 DNA ligase(“ligase”) per 0.5 μg of approximately equimolar amounts of the DNAfragments to be ligated.

[0091] “Filling” or “blunting” refers to the procedures by which thesingle stranded end in the cohesive terminus of a restrictionenzyme-cleaved nucleic acid is converted to a double strand. Thiseliminates the cohesive terminus and forms a blunt end. This process isa versatile tool for converting a restriction cut end that may becohesive with the ends created by only one or a few other restrictionenzymes into a terminus compatible with any blunt-cutting restrictionendonuclease or other filled cohesive terminus. Typically, blunting isaccomplished by incubating 2-15 μg of the target DNA in 10 mM MgCl₂, 1mM dithiothreitol, 50 mM NaCl, 10 mM Tris (pH 7.5) buffer at about 37°C. in the presence of 8 units of the Klenow fragment of DNA polymerase 1and 250 μM of each of the four deoxynucleoside triphosphates. Theincubation generally is terminated after 30 min. phenol and chloroformextraction and ethanol precipitation.

EXAMPLE 1 Cloning of Human Pancreatic DNase I

[0092] cDNA Library Preparation

[0093] A human pancreatic cDNA library was constructed in λgt10 usingpolyadenylated mRNA prepared from freshly obtained and liquid N₂ frozenhuman pancreas (Lauffer et al., Nature 318:334 [1985]). Using oligo dTprimers and EcoRI-SalI-XhoI-SstII adapters, a cDNA library of 0.9×10⁶independent isolates of greater than 600 bp was obtained.

[0094] Oligonucleotide Probes

[0095] Two long probes were synthesized based on the amino acid sequenceof bovine DNase I. Two segments of amino acid sequence which exhibitedlow redundancy were selected and mammalian codon usage tables wereemployed.

[0096] Probe 1:5′GTG-CTG-GAC-ACC-TAC-CAG-TAT-GAT-GAT-GGC-TGT-GAG-TCC-TGT-GGC-AAT-GAC 3′(51 mer corresponding to the amino sequenceVal-Leu-Asp-Thr-Tyr-Gln-Tyr-Asp-Asp-Gly-Cys-Glu-Ser-Cys-Gly-Asn-Asp)

[0097] Probe 2:5′TAT-GAC-GTC-TAC-CTG-GAC-GTG-CAG-CAG-AAG-TGG-CAT-CTG-AAT-GAT-GTG-ATG-CTG-ATG-GGC-GAC-TTC-AAC-GC3′ (71 mer corresponding to the amino acid sequenceTyr-Asp-Val-Tyr-Leu-Asp-Val-Gln-Gln-Lys-Trp-His-Leu-Asn-Asp-Val-Met-Leu-Met-Gly-Asp-Phe-Asn)

[0098] Isolation of Human DNase I cDNA Clones

[0099] The two probes were end-labeled with T4 polynucleotide kinase and[³²P]adenosine triphosphate (Maniatis et al., Molecular Cloning, [ColdSpring Harbor Laboratory, 1982]), and used separately to screen thehuman pancreatic cDNA library under low stringency hybridizationconditions: 20% formamide, 5× SSC (0.75 M NaCl, 0.075 M sodium citrate),50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS,and 10% dextran sulfate at 42° C. Low stringency washes were carried outin 1× SSC and 0.1% SDS at 42° C. Three (1, 2, and 6) of 600,000 cloneshybridized with both probes and contained 1.3 kB inserts. These weresubcloned into M13 vectors (Messing et al., Nucleic Acids Res. 9:309[1981]) and sequenced by the chain-termination method (Sanger et al., J.Mol. Biol. 143:161 [1980]).

[0100] Comparison of the deduced amino acid sequence of clone 6 with theamino acid sequence of bovine pancreatic DNase I revealed extensivehomology. Notable, however, was a large deletion and a large insertionin the deduced amino acid sequence of clone 6. In addition the insertioncontained a stop at its termination and had the characteristics of aretained intron due to a misspliced message. Clones 1 and 2 alsocontained the putative intron.

[0101] Two additional exact nucleotide probes from the sequence of clone6 were synthesized for obtaining additional clones.

[0102] Probe 4 (N-terminal probe): 5′CTG-AAG-ATC-GCA-GCC-TTC-AAC-ATC-CAG-ACA-TTT-GGG-GAG-ACC (42 mer)

[0103] Probe 5 (putative intron probe):5′TCC-GCA-TGT-CCC-AGG-GCC-ACA-GGC-AGC-GTT-TCC-TGG-TAG-GAC (42 mer)

[0104] The probes were labeled with ³²P and used to rescreen 1.3×10⁶clones from the human pancreatic cDNA library at high stringency: 50%formamide, 5× SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt's, sonicatedsalmon sperm DNA (50 g/ml), 0.1% SDS, and 10% dextran sulfate at 42° C.Washes were carried out at 42° C. in 0.2× SSC and 0.1% SDS. Four cloneshybridized with probe 4 (N-terminal probe) and not with probe 5(putative intron probe). Of these, clone 18-1, which contained an insertof approximately 1.1 kB by PAGE, was subcloned into M13 and sequenced.The full nucleotide sequence of clone 18-1 and the deduced amino acidsequence is shown in FIG. 1.

[0105] Clone 18-1 is composed of 1039 bp and has a long open readingframe without the intron found in clone 6. As noted, two possibleinitiation codons (ATG) are present (positions 160-162 and 169-171).Either may be used. The former ATG with a purine at position -3 moreclosely conforms to the Kozak rule (Kozak, Cell 44:283 [1986]). Betweenthe putative initiation codons and the nucleotides corresponding to theN-terminal leucine (CTG at position 226-228 are nucleotides encoding ashort, relatively hydrophobic amino acid sequence (19-22 amino acids inlength) which is a likely secretion signal sequence.

[0106] Comparison of the deduced amino acid sequence of human DNase Iwith bovine DNase I reveals extensive amino acid homology (FIG. 2). Thehuman and bovine proteins are 260 aa in length and possess an N-terminalleucine. The major structural features of the bovine protein, whichinclude the four cysteines (101, 104, 173 and 209), the two potentialN-linked glycosylation sites (18 and 106), and the active site histidine(134), are conserved. Overall there is approximately 77% amino acididentity.

[0107] The six regions of greatest amino acid diversity (amino acidresidues 27-39, 83-96, 115-126, 156-161, 219-234 and 243-247) eachcontain more than three differences and more than 54% of the amino acidsare variable. Interestingly, these regions are also variable when thebovine sequence is compared with the ovine and porcine. Some of theseregions are exposed loop structures according to the Xray crystalstructure of bovine DNase (Oefner et al., J. Mol. Biol. 192:605 [1986]),and thus would be predicted to be relatively immunogenic. Thus, it islikely that the amino acid differences between the human and the bovinesequences may lead to immunologic reactions.

EXAMPLE 2 Assays for DNase Activity

[0108] In addition to standard ELISA (see example 4) and RIA, threeassays have been developed to detect DNase activity.

[0109] 1. Hydrolysis of ³²P-labeled DNA. Radiolabeled ³²P-DNA wasprepared using an M13 single-stranded template, a 17mer sequencingprimer, ³²P-dCTP, non-radioactive dATP, dTTP, and dGTP, and Klenow.Briefly, 1.5λ MgCl2 (35 mM), 1.5λ 10× restriction buffer (70 mMTris-HCl, pH 7.6; 35 mM dithiothreitol; 1 mM EDTA), and 6λ H₂O weremixed with 1λ template (approximately 1 μg) and 1.5λ Palmer (0.5 μM) andheated to 55° C. for 7 min. Nucleotide mix was prepared by taking 40λ³²P-dCTP (sp. act.3000 Ci/mmol; 400 μCi) plus 1λ each of 2 mM stocks ofnon-radioactive dATP, dTTP, and dGTP to dryness and then reconstitutingthe nucleotides in 7λ 1× restriction buffer. The nucleotide mix and 1λKlenow were added to the template-primer mixture and the reaction wasincubated at 37° C. After 15 min, 2λ of a non-radioactivedeoxynucleotides were added and the incubation was continued for anadditional 15 min. Radiolabeled DNA was then separated from freenucleotides by centrifugation through Sephadex G-50 (Maniatis et al.,[1982]).

[0110] DNase activity is measured by incubating samples with 100,000 cpm³²P-DNA plus 80 μg/ml non-radioactive salmon sperm DNA in DNase buffer(10 mM Tris-HCl pH 7, 4 mM MgCl2, 4 mM CaCl₂) for 45 min at 37° C. Thereaction is terminated by the addition of one-half volume ofnon-radioactive DNA (2 mg/ml) and one volume of ice-cold 20%trichloroacetic acid. After 10 min at 4° C., the mixture is centrifugedat 12,000 g×10 min, and an aliquot of the supernatant is counted. Thepresence of acid-soluble counts in the supernatant reflects DNaseactivity. Each day a standard curve is generated by testing 0.1-200 ngof bovine DNase I (Sigma D-5025).

[0111] 2. Agar plate DNase assay. Smith, Hankock, and Rhoden (AppliedMicrobiology, 18:991 [1969]) described a test agar containing methylgreen and DNA for determining DNase activity of microorganisms. Thisassay was modified in order to develop a rapid semi-quantitative assayfor soluble DNase activity in order to screen cell supernatants tomonitor expression and to screen column fractions to monitorpurification. To prepare the agar, bacto agar (7.5 g) is melted in 500ml of buffer (25 mM Tris pH 7.5, 5 mM MgCl2, 5 mM CaCl₂, 0.1% sodiumazide). Salmon sperm DNA (1.0 g) and methyl green (17.5 mg) are added.After stirring for 1-2 hr at 55° C., and autoclaving, plates are pouredand stored at 4° C. DNase activity is measured by spotting 0.5-5λaliquots onto the plates, which are then incubated at room temperatureor 37° C. for 4-48 hr. A standard curve is generated by aliquoting0.1-1000 ng of bovine DNase I. DNase is readily measured by the size ofthe clear zones in the agar, there being a logarithmic relationshipbetween DNase concentration and diameter of the clear zones. This assayformat also is applicable for the rapid identification ofhigh-expressing DNase-producing clones, either with a view towardsproducing DNase or towards screening transformants in which the DNase isused as a selectable marker or as a reporter gene.

[0112] In addition to using methyl green and DNA in an agar plateformat, these reagents are also used in an aqueous format (e.g. in96-well plates) to rapidly, sensitively, and specifically quantitateDNase activity.

[0113] 3. SDS-polyacrylamide gel electrophoresis and xymography.SDS-polyacrylamide gel electrophoresis and xymography was performed by amodification of the procedure of Rosenthal and Lacks (Anal. Biochem.80:76 [1977]). Briefly, the buffer system of Laemmli was used to prepare12% polyacrylamide gels. Salmon sperm DNA (10 μg/ml) and EDTA (2 mM)were added to both the stacking and the resolving gels prior topolymerization. Proteins were suspended in sample buffer, heated at 100°C. for 3 min, prior to application to the gels. Electrophoresis wasconducted at room temperature at constant current. Followingelectrophoresis, the gel was rinsed with water and incubated in 250 mlof 40 mM Tris-HCl, pH 7.5, 2 mM MgCL₂, 0.02% azide. After 1 hr, freshbuffer was added and the gel was soaked for 12 hr at 24° C. To revealDNase activity, the gel was put into fresh buffer containing 2 mM CaCl₂and 1 μg/ml ethidium bromide, and then examined under short-wave UVlight at intervals from 5 min to 24 hr. To stop the reaction EDTA isadded (final concentration 15 mM), and the xymogram is photographed. Thegel can then be stained for protein by Coomassie blue.

EXAMPLE 3 Expression of Human DNase I

[0114] 1. nRK.DNase.7

[0115] Plasmid pRK.DNase.7 was constructed from clone 18-1, describedabove, as follows:

[0116] The plasmid pRK5 was digested with EcoRI, dephosphorylated, andfragment 1 comprising the bulk of the plasmid was isolated. pRK5 isdescribed in Suva et al., Science 237:896 (1987); U.S. Ser. No. 97,472,filed Sep. 11, 1987; and EP Publ. 307,247, published Mar. 15, 1989,where the pCIS2.8c28D starting plasmid is described in EP 278,776published Aug. 17, 1988 based on U.S. Ser. Nos. 07/071,674 and06/907,297. The A DNase clone 18-1 was digested with EcoRI and theinsert (fragment 2) was isolated. Fragment 1 and fragment 2 were ligatedand the ligation mixture transformed into E. coli strain 294. Thetransformed culture was plated on ampicillin media plates and resistantcolonies selected. Plasmid DNA was prepared from transformants andchecked by restriction analysis for the presence of the correctfragment.

[0117] pRK.DNase.7 was transfected into human embryonic kidney-293 cellsfor transient and stable expression. For transient expression of humanDNase I, 60 mm plates of confluent HEK-293 cells (50%) were transfectedas previously described (Eaton et al. 1986) by the calcium phosphatemethod (Wigler et al., 1979). For stable expression of human DNase I,HEK-293 cells were similarly transfected simultaneously with pRK.DNase.7and a plasmid expressing the neomycin resistance gene pRSV neo (Gormanet al., 1985). Two days after transfection, the cells were passaged intostandard medium (1:1 F12/DME supplemented with L-glutamine,penicillin-streptomycin and 10% FBS) with 0.5 mg/ml G418 (Genticinsulfate; Gibco) for selection of stable lines.

[0118] Analysis was undertaken of supernatants of 293 cells transfectedeither transiently or stably with the DNase plasmid revealed 0.2-1.0μg/ml of DNase activity, as measured either by the ³²P-DNA hydrolysisassay or the green agar plate DNase assay. Analysis of the transfectedcell supernatants by SDS-PAGE and xymography revealed a new protein bandat approximately 35-37 kD with DNase activity. Additional studies showedthe recombinant human DNase I produced in 293 cells required calcium foractivity, was inhibited by EDTA, heat, and actin, and had greateractivity for double stranded DNA than for single stranded DNA. Thespecific activity of human DNase expressed in 293 cells appearedcomparable to that of bovine DNase.

[0119] 2. pSVeDNaseDHFR3

[0120] pSVeDNAseDHFR3, a plasmid suitable for recombinant synthesis ofhuman DNase I in CHO cells was constructed as follows:

[0121] An intermediate plasmid was constructed in order to remove aredundant polylinker region. Plasmid pRK.DNase.7 was digested with EcoRIand SphI and the largest fragment containing the 5′ portion of the DNasecoding region was isolated (fragment 3). Plasmid pRK.DNase.7 wasdigested with SalI, blunted with Klenow, digested with SphI, and theintermediate size fragment containing the 3′ portion of the DNase codingregion was isolated (fragment 4). pRK5 was cut with SmaI and EcoRI andthe fragment comprising the bulk of the plasmid was isolated (fragment5). Fragments 3, 4 and 5 were ligated and the mixture was transformedinto E. coli strain 294. The transformed culture was plated onampicillin media plates and resistant colonies selected. Plasmid DNA wasprepared from transformants and checked by restriction analysis for thepresence of the correct fragment. The resulting plasmid is referred toas pRKDNaseInt.

[0122] Plasmid pE342HBV.E400D22 (Crowley et al., “Mol. Cell Biol.” 3:44[1983]) was digested with EcoRI and PvuI and the smallest fragmentcontaining the SV40 early promoter and part of the β-lactamase gene wasisolated (fragment 5). Plasmid pE342HBV.E400D22 was also digested withBamHI and PvuI and the fragment comprising the bulk of the plasmidcontaining the balance of the β-lactamase gene as well as the SV40 earlypromoter and the DHFR gene was isolated (fragment 6). PlasmidpRKDNaseInt was digested with EcoRI and BamHI and the DNase codingfragment was isolated (fragment 7). Fragments 5, 6, and 7 were ligatedand the mixture was transformed into E. coli strain 294. The transformedculture was plated on ampicillin media plates and resistant coloniesselected. Plasmid DNA was prepared from transformants and checked byrestriction analysis for the presence of the correct fragment. Theresulting plasmid is referred to as pSVeDNaseDHFR3.

[0123] For stable expression of human DNase I, using the above plasmid,60 mm plates of confluent CHO cells (DP-7) were transfected by thecalcium phosphate method and grown initially in selective medium.Unamplified cell lines have grown out whose medium containsapproximately 0.02-0.1 μg/ml of DNase activity, as measured either bythe ³²P-DNA hydrolysis assay or the green agar plate DNase assay. It isanticipated that higher levels (at least 5×) of expression would beachieved if these cells were grown to high density in a fermentor.Individual clones would be selected for their individual levels of DNaseexpression to pick high secreting cells, and thereafter amplifying eachselected clone in the presence of increasing concentrations of MTX (12.5to 2000 nM)

[0124] 3. pDNA11

[0125] A plasmid was constructed suitable for recombinant synthesis ofDNase in E. coli as a secreted protein. This plasmid is called pDNA11and was constructed as follows.

[0126] Plasmid pTF.III (U.S. Ser. No. 07/152,698) was digested with NsiIand SalI and the largest fragment comprising the bulk of the plasmid wasisolated (fragment 8). Plasmid pRKDNase7 was digested with SalI andBstXI and the 798 bp fragment comprising most of the coding region wasisolated (fragment 9). Two synthetic oligonucleotides were synthesized:

[0127] DLink 1: 5′TTG-AAG-ATC-GCA-GCC-TTC-AAC-ATC-CAG-ACA-T (31mer)

[0128] DLink 3: 5′CTG-GAT-GTT-GAA-GGV-TGC-GAT-CTT-CAA-TGC-A (31mer)

[0129] Fragments 8 and 9 and synthetic oligonucleotides DLink 1 andDLink 3 were ligated and the mixture was transformed into E. coli strain294. The transformed culture was plated on ampicillin media plates andresistant colonies selected. Plasmid DNA was prepared from transformantsand checked by restriction analysis for the presence of the correctfragment and preservation of the NsiI and BstXI restriction sites.Several plasmids were sequenced to confirm incorporation of correctsynthetic DNA. 294 cells transformed with pDNA11 expressed >500 mg/L oftwo new major proteins as revealed by SDS-PAGE—a major band atapproximately 32 kD and a minor band at approximately 30 kD. Amino acidsequence analysis of the two bands revealed N-terminal sequences ofMet-Lys-Lys-Asn-Ile-Ala and Leu-Lys-Ile-Ala-Ala-Phe, respectively. Thus,the higher MW band represents unprocessed human DNase and the lower MWband represents properly processed native human DNase.

[0130] Human DNase expressed in E. coli is active. 294 cells transformedwith pDNA11 grown on agar plates supplemented with calcium, magnesium,and low phosphate revealed secretion of active DNase, as evidenced thepresence of a clear zone surrounding the transformed cells, and notcontrol cells. In addition, transformed cells solubilized with SDS andbeta-mercaptoethanol were run into SDS-PAGE gels and xymography wasperformed. DNase activity was associated with the band of properlyprocessed human DNase, but not with unprocessed human DNase.

[0131] 4. pDNA2

[0132] A plasmid was constructed for recombinant expression of humanDNase I as an intracellular protein in E. coli. The plasmid is calledpDNA2, and was constructed as follows.

[0133] Plasmid pHGH207/307 was prepared from pHGH207 (U.S. Pat. No.4,663,283) by removing the EcoRI site upstream from the trp promoter(EcoRI digested, blunted and religated).

[0134] Plasmid pHGH207/307 was digested with XbaI and SalI and thelargest fragment comprising the bulk of the plasmid was isolated(fragment 10). Plasmid pRK.DNase.7 was digested with SalI and BstXI andthe 798 bp fragment comprising most of the coding region was isolated(fragment 9). Two synthetic oligonucleotides were synthesized:

[0135] DLink 4: 5′CTAGAATTATG-TTA-AAA-ATT-GCA-GCA-TTT-AAT-ATT-CAA-ACA-T(42mer)

[0136] DLink 5: 5′TTG-AAT-ATT-AAA-TGC-TGC-AAT-TTT-TAACATAATT (34mer)

[0137] Fragments 9 and 10 and synthetic oligonucleotides DLink 4 andDLink 5 were ligated and the mixture was transformed into E. coli strain294. The transformed culture was plated on ampicillin media plates andresistant colonies selected. Plasmid DNA was prepared from transformantsand checked by restriction analysis for the presence of the correctfragment and preservation of the XbaI and BstXI restriction sites.Several plasmids were sequenced to confirm incorporation of correctsynthetic DNA.

[0138] 294 cells transformed with pDNA2 expressed one new major proteinas revealed by SDS-PAGE of approximately 30 kD. Amino acid sequenceanalysis of protein revealed N-terminal sequenceMet-Leu-Lys-Ile-Ala-Ala-Phe, corresponding to human Met-DNase.

[0139] Human Met-DNase expressed directly in E. coli is also active.Transformed cells solubilized with SDS and beta-mercaptoethanol were runinto SDS-PAGE gels and xymography was performed. DNase activity wasassociated with the band of human Met-DNase.

EXAMPLE 4 Further Analysis of DNase Expression

[0140] a. DNase Expression Vectors

[0141] The human pancreatic deoxyribonuclease (DNase) I cDNA fromplasmid pRK.DNase.7 described above, which contains the entire insertfrom DNase cDNA clone 18-1 isolated from a λgt10 human pancreatic cDNAlibrary in vector pRK5, was transferred into pRK5 to create theintermediate expression vector pRK.DNase.3. In this plasmid(pRK.DNase.3), DNase synthesis is directed by the CMV transcriptionregulatory elements. A splice unit, described below, is located betweenthe transcription and translation initiation sites. For generation ofDNAse expression vectors, the CMV transcription regulatory sequences andthe splice unit of pRK.DNase.3 were replaced by the SV40 transcriptionregulatory sequences and different splice donor-intron-splice acceptorunits as described below. For comparison, a corresponding vector lackingthe splice unit was also created (pSve.DNase).

[0142] 1. pRK.DNase.3

[0143] Vector pRK.DNase.3 (FIG. 3) was constructed as follows: pRK5 wasdigested with SmaI and SalI, which cut exclusively in the polylinkerregion between the 5′ and 3′ control sequences, and the large fragmentwas isolated. The pRK5 vector contains a splice donor-intron-spliceacceptor region upstream of a coding region and downstream of apromoter, where the intron region consists of a cytomegalovirus (CMV)immediate early splice donor and intron sequences, a bacteriophage SP6promoter insert, and immunoglobulin (Ig) heavy chain variable region(V_(H)) intron and splice acceptor sequences. Vector pRK.DNase.7 wascleaved with BsmI, the 3′ protruding ends were trimmed back with T4 DNApolymerase, and the material was redigested with SalI to release theentire human DNase I coding sequence as a 921-nt fragment. After gelisolation, this fragment was ligated to the pRK5 large fragment usingstandard ligation methodology (Maniatis et al. , 1982, supra) to createvector pRK.DNase.3.

[0144] pRK.DNase.3 contains CMV transcription regulatory elements, and asplice unit located between the transcription and translation initiationsites (“Intron” in FIG. 3). In this vector, the splice unit of the pRK5vector is present without any modifications.

[0145] 2. pSVe.DNase

[0146] Vector pSVe.DNase was constructed as follows (FIG. 3): Theregulatory sequences preceding the DNAse coding region in vectorpRK.DNase.3 were separated from the remainder of the vector by digestionwith SstI and ClaI; DNA prepared from dam+bacterial host cells was usedto prevent cleavage of the second ClaI site in the vector, locatedtowards the 3′ end of the SV40 early polyadenylation region. The largestfragment lacking the 5′ control region was gel isolated.

[0147] DHFR expression vector pE348DHFRUC served as a source of the SV40transcription regulatory sequences. The vector pE348DHFRUC (Vannice andLevinson, J. Virology, 62:1305-1313, 1988, where it is designated pE,FIG. 1) contains the SV40 enhancer and early promoter region upstream ofthe HindIII site, including the SV40 early sties of transcriptioninitiation (position 5171 in the virus), preceding cDNA encoding murinedihydrofolate reductase (DHFR), which is followed by the 584-bpHepatitis B virus (HBV) polyA signal in plasmid pMLl from the GamHI tothe BGlII sites of HBV. This plasmid contains a polylinker immediatelyupstream of the SV40 sequences. The SV40 transcription regulatorysequences, were released by digestion with SstI and ClaI, and theresulting 5′ protruding ends were filled in using Klenow poll in thepresence of all four deoxyribonucleotides (dNTPs: dATP, dGTP, dCTP,TTP). Upon subsequent digestion with XbaI, the SV40 transcriptionregulatory sequences (enhancer and early promoter, including the SV40early sites of transcription initiation), present on the smallerXbaI-ClaI fragment (360 nucleotides) were gel isolated.

[0148] The smaller fragment from pE34DHFRUC described immediately abovewas gel isolated and ligated to the large pRK.DNase.3 fragment togenerate vector pSVe.DNase. In this vector, DNase synthesis is directedby the SV40 transcription control sequences, but no splice unit ispresent between these and the DNase coding region.

[0149] 3. pSVI.DNase

[0150] Vector pSVI.DNase (FIG. 4) was prepared after insertion of theDNase coding sequence into an intermediate plasmid, pRK5.5Ve. Thisintermediate was created by replacing the pRK5 CMV transcriptionregulatory elements, located between SpeI and SacII restriction sites,with the small XbaI-HindIII fragment from pE348DHFRUC that contains theSV40 early promoter and enhancer. The 3′ protruding ends generated bySacII digestion of pRK5 were chewed back with T4 DNA polymerase, and the5′ protruding ends resulting from HindIII digestion of pE348DHFRUC werefilled in using T4 polymerase in the presence of all four dNTPs.

[0151] For construction of pSVI.DNase, the DNase coding sequence wasisolated from vector pRK.DNase.3 by cleavage with EcoRI and HindIII, andinserted into the large fragment of pRK5.Sve that had been isolatedafter digestion with the same two enzymes. Vector pSVI.DNase containsthe SV40 transcription regulatory elements (enhancer and early promoter,including the SV40 early sites of transcription initiation) and mRNA capsites, followed by the splice-donor-intron-splice acceptor unit of thepRK5 vector without any modifications, the cDNA encoding DNase, the SV40early polyadenylation (“polyA”) region, and the SV40 origin ofreplication (“ori”) regions from SV40.

[0152] The complete nucleotide sequence of pSVI.DNase up to, but notincluding, the coding region of DNase is shown in FIG. 6.

[0153] 4. pSVI2.DNase. pSVI3.DNase. pSVI5.DNase. and pSVI6B.DNase

[0154] Vectors pSVI2.DNase, pSVI3.DNase, pSVI5.DNase, and pSVI6B.DNasewere constructed (FIG. 5) by recombining two fragments in each case. Thefirst was the large pRK.DNase.3 fragment resulting from digestion withEcoRI, treatment with T4 DNA polymerase in the presence of dNTPs, andsubsequent cleavage with SstI; the second was in each case the smallfragment containing the SV40 5′ regulatory sequences and a modifiedsplice unit.

[0155] Modifications to splice units for enhancing recombinantexpression in mammalian cells are described in a copending applicationfiled Nov. 3, 1989, given U.S. Ser. No. ______, specificallyincorporated herein by reference. FIG. 11 shows a schematicrepresentation of the splice unit nucleotide sequences involved in thepreparation of the vectors of this example, i.e., SVI, SVI2, SVI3, SVI5,and SVI6B. The boxes represent changes from the SVI sequence, the doubleunderlining is a spurious ATG codon, the underlining shows spurioussplice sites and added or changed branchpoint sequence (BPS) regions,the breaks in sequence represent deletions of the nucleotides forSVI3-SVI5, the designation indicates sequence not shown, and the caretsindicate the 5′ and 3′ cleavage sites within the splice donor and spliceacceptor, respectively, of the the splice unit. These sequences may beprepared by known methods of protein synthesis, or by standardmodifications of the pSVI sequence, or by the method of U.S. Ser. No.______, id..

[0156] FIGS. 7-10 show the complete nucleotide sequences of PSVI2.DNase,pSVI3.DNase, pSVI5.DNase, and pSVI6B.DNase, respectively, up to but notincluding the coding region of DNase. The splice unit sequences of FIG.11 are incorporated in FIGS. 7-10.

[0157] b. Transient DNase Expression

[0158] Transient expression directed by the different DNase vectors wasanalyzed after transfection into CHO dhfr⁻ cells. Cells were transfectedby a modification of the DEAE-dextran procedure and levels of DNaseaccumulated in the cell media 36 to 48 hours after transfection.Approximately 4×10₅ cells were plated per well in six-well 35 mm culturedishes. The following day, the volume of 2 μg DNase expression vectorwas adjusted to 15 μl by the addition of TBS (137 mM NaCl, 5 mM KCl, 1.4mM Na₂PO₄, 24.7 mM TrisHCl, 1.35 mM CaCl₂, 1.05 mM MgCl₂, pH 7.5). 30 μlDEAE-dextran (10/mg/ml) and 2 ml serum-free culture medium containing100 μM chloroquine were then added. The cell media was removed, thecells were rinsed once with PBS (phosphate buffered saline, pH 7.5), andthe DNA mixture was added. This was followed two to three hours later bya glycerol shock, and the cells were covered with 2 ml regular growthmedia until assayed. The DNase vectors were cotransfected with 2 μg of acontrol plasmid, pRSV.hGH, which directs expression of the human growthhormone (hGH) gene controlled by the transcription regulatory sequencesin the long terminal repeat (LTR) of Rous sarcoma virus. This plasmidmay be prepared by replacing the ras promoter of rasP.hGH described inCohen and Levinson, Nature, 334: 119-124 (1988) with the RSV promoter.The amount of hGH synthesized in each case served as a standard to allowfor a more precise comparison of the DNase levels obtained in thedifferent transfections. Shown below are the levels of DNase and hGHproduced in a typical experiment performed in duplicate: (corrected)ng/ml DNase ng/ml hGH ng/ml DNase Vector run 1 run 2 run 1 run 2 run 1run 2 pSVe.DNase 19.7 17.4 50.1 42.0 8.6 9.2 pSVI.DNase 17.9 13.8 29.720.4 12.8 14.7 pSVI2.DNase 34.2 30.5 41.9 40.3 18.0 17.0 pSVI3.DNase37.8 28.7 45.2 40.5 18.0 15.1 pSVI5.DNase 28.4 22.1 37.2 28.7 16.7 17.0pSVI6B.DNase 28.5 37.8 61.3 67.5 10.2 12.2

[0159] DNase levels in the media were measured by a standard ELISA usingserum from rabbits injected either with human DNase or bovine DNase andadjuvant (Practice and Theory of Enzyme Immunoassays, P.Tijssen, Chapter5, “Production of antibodies”, pg. 43-78 (Elsevier, Amsterdam 1985)).hGH levels were determined using a commercially available assay kit(IRMA, immunoradiometric assay) purchased from Hybritech, Inc., LaJolla, Calif.

[0160] The data in the last column suggest that DNase expressiondirected by vectors pSVI2.DNase, pSVI3.DNase, and pSVI5.DNase issomewhat higher than that obtained with the parental vector pSVI.DNase.The numbers in the first column show more significant differences thatnow apply also to pSVI6B.DNase. Although it would appear that the thirdcolumn presents the more credible set of data, it is not certain thatefficient hGH synthesis does not adversely affect the level of DNasesynthesis. For example, without limitation to any one theory, expressionat this level may likely cause competition for components in thesecretory pathway, and as a result a reduced DNase level when hGHexpression is high. Other competitive effects may also bias the resultssuch that the first column of data may well represent the actualdifferences in expression capability among the various DNase vectors. Ineither case, it is clear that at least some of the vectors containing amodified splice unit express higher levels of DNase in a transient assaythan the corresponding vector that has the original splice unit.

[0161] c. Stable DNase Expression

[0162] 2 μg of plasmid pSVe.DNase, pSVI.DNase, pSVI2.DNase, pSVI3.DNase,pSVI5.DNase, or pSVI6B.DNase were transfected into CHO-dhfr⁻ cells in a60-mm dish with 0.1 μg of pFD11. After two days the cells were split 90%and 10% in 10-cm dishes. When colonies appeared, they were counted andassayed as mixed colonies. Per cell assays were performed in duplicate(designated A or B in the table below), except for pSVI2.DNase which wasmeasured once. In this assay, the cell line was set up at 2×10⁵cells/well-6 well dish in 3 ml. Three days later cells were counted andthe media was assayed for DNase using the DNase ELISA described abovewith a range of 0.2 to 25 ng/ml. The results are indicated below:pg/cell/day DNase Vector A B pSVe.DNase 0.057 0.040 pSVI.DNase 0.0790.016 pSVI2.DNase 0.067 pSVI3.DNase 0.048 0.043 pSVI5.DNase 0.014 0.005pSVI6B.DNase 0.039 0.062

EXAMPLE 5 Reduction in Viscosity of Purulent Sputum by Recombinant HumanDNase

[0163] The effects of recombinant human DNase I and pure bovine DNase I(Worthington) on purulent sputum produced by a patient with cysticfibrosis was examined using a simple pourability assay. Briefly, sampleswere incubated with approximately 100 μl of sputum in Eppendorf testtubes. After various periods of time at 37° C., the tubes were invertedand the ability of the sputum to pour down the side of a test tube wasevaluated on a scale of 0 (no movement) to 5+(free-flowing down the sideof the tube). Whereas 293 supernatants from human DNase I transfectedcells caused a change of 4-5+after 30 min incubation, untransfected cellsupernatants had no effect. The specificity was confirmed by showingthat EDTA—which inhibits bovine and human DNase—completely prevented the293 supernatants from human DNase I transfected cells from liquefyingcystic fibrosis sputum. In addition, the effects on sputum of bovineDNase which was pure and free of proteases was examined for the firsttime. Previously published reports all utilized bovine DNase whichacknowledged to be contaminated with significant quantities ofchymotrypsin and trypsin, proteins which had been shown to have activityon sputum by themselves. Pure bovine DNase I, free of proteases, rapidlyliquified purulent sputum. Thus, pure DNase alone is effective atreducing the viscosity of sputum.

1 25 51 base pairs Nucleic Acid Single Linear 1 GTGCTGGACA CCTACCAGTATGATGATGGC TGTGAGTCCT GTGGCAATGA 50 C 51 17 amino acids Amino AcidLinear 2 Val Leu Asp Thr Tyr Gln Tyr Asp Asp Gly Cys Glu Ser Cys Gly 1 510 15 Asn Asp 71 base pairs Nucleic Acid Single Linear 3 TATGACGTCTACCTGGACGT GCAGCAGAAG TGGCATCTGA ATGATGTGAT 50 GCTGATGGGC GACTTCAACG C71 23 amino acids Amino Acid Linear 4 Tyr Asp Val Tyr Leu Asp Val GlnGln Lys Trp His Leu Asn Asp 1 5 10 15 Val Met Leu Met Gly Asp Phe Asn 2042 base pairs Nucleic Acid Single Linear 5 CTGAAGATCG CAGCCTTCAACATCCAGACA TTTGGGGAGA CC 42 42 base pairs Nucleic Acid Single Linear 6TCCGCATGTC CCAGGGCCAC AGGCAGCGTT TCCTGGTAGG AC 42 31 base pairs NucleicAcid Single Linear 7 TTGAAGATCG CAGCCTTCAA CATCCAGACA T 31 31 base pairsNucleic Acid Single Linear 8 CTGGATGTTG AAGGVTGCGA TCTTCAATGC A 31 42base pairs Nucleic Acid Single Linear 9 CTAGAATTAT GTTAAAAATT GCAGCATTTAATATTCAAAC AT 42 34 base pairs Nucleic Acid Single Linear 10 TTGAATATTAAATGCTGCAA TTTTTAACAT AATT 34 7 amino acids Amino Acid Linear 11 Met LeuLys Ile Ala Ala Phe 1 5 1039 base pairs Nucleic Acid Double Linear 12TCCTGCACAG GCAGTGCCTT GAAGTGCTTC TTCAGAGACC TTTCTTCATA 50 GACTACTTTTTTTTCTTTAA GCAGCAAAAG GAGAAAATTG TCATCAAAGG 100 ATATTCCAGA TTCTTGACAGCATTCTCGTC ATCTCTGAGG ACATCACCAT 150 CATCTCAGGA TGAGGGGCAT GAAGCTGCTGGGGGCGCTGC TGGCACTGGC 200 GGCCCTACTG CAGGGGGCCG TGTCCCTGAA GATCGCAGCCTTCAACATCC 250 AGACATTTGG GGAGACCAAG ATGTCCAATG CCACCCTCGT CAGCTACATT300 GTGCAGATCC TGAGCCGCTA TGACATCGCC CTGGTCCAGG AGGTCAGAGA 350CAGCCACCTG ACTGCCGTGG GGAAGCTGCT GGACAACCTC AATCAGGATG 400 CACCAGACACCTATCACTAC GTGGTCAGTG AGCCACTGGG ACGGAACAGC 450 TATAAGGAGC GCTACCTGTTCGTGTACAGG CCTGACCAGG TGTCTGCGGT 500 GGACAGCTAC TACTACGATG ATGGCTGCGAGCCCTGCGGG AACGACACCT 550 TCAACCGAGA GCCAGCCATT GTCAGGTTCT TCTCCCGGTTCACAGAGGTC 600 AGGGAGTTTG CCATTGTTCC CCTGCATGCG GCCCCGGGGG ACGCAGTAGC650 CGAGATCGAC GCTCTCTATG ACGTCTACCT GGATGTCCAA GAGAAATGGG 700GCTTGGAGGA CGTCATGTTG ATGGGCGACT TCAATGCGGG CTGCAGCTAT 750 GTGAGACCCTCCCAGTGGTC ATCCATCCGC CTGTGGACAA GCCCCACCTT 800 CCAGTGGCTG ATCCCCGACAGCGCTGACAC CACAGCTACA CCCACGCACT 850 GTGCCTATGA CAGGATCGTG GTTGCAGGGATGCTGCTCCG AGGCGCCGTT 900 GTTCCCGACT CGGCTCTTCC CTTTAACTTC CAGGCTGCCTATGGCCTGAG 950 TGACCAACTG GCCCAAGCCA TCAGTGACCA CTATCCAGTG GAGGTGATGC1000 TGAAGTGAGC AGCCCCTCCC CACACCAGTT GAACTGCAG 1039 341 amino acidsAmino Acid Linear 13 Ser Cys Thr Gly Ser Ala Leu Lys Cys Phe Phe Arg AspLeu Ser 1 5 10 15 Ser Thr Thr Phe Phe Ser Leu Ser Ser Lys Arg Arg LysLeu Ser 20 25 30 Ser Lys Asp Ile Pro Asp Ser Gln His Ser Arg His Leu GlyHis 35 40 45 His His His Leu Arg Met Arg Gly Met Lys Leu Leu Gly Ala Leu50 55 60 Leu Ala Leu Ala Ala Leu Leu Gln Gly Ala Val Ser Leu Lys Ile 6570 75 Ala Ala Phe Asn Ile Gln Thr Phe Gly Glu Thr Lys Met Ser Asn 80 8590 Ala Thr Leu Val Ser Tyr Ile Val Gln Ile Leu Ser Arg Tyr Asp 95 100105 Ile Ala Leu Val Gln Glu Val Arg Asp Ser His Leu Thr Ala Val 110 115120 Gly Lys Leu Leu Asp Asn Leu Asn Gln Asp Ala Pro Asp Thr Tyr 125 130135 His Tyr Val Val Ser Glu Pro Leu Gly Arg Asn Ser Tyr Lys Glu 140 145150 Arg Tyr Leu Phe Val Tyr Arg Pro Asp Gln Val Ser Ala Val Asp 155 160165 Ser Tyr Tyr Tyr Asp Asp Gly Cys Glu Pro Cys Gly Asn Asp Thr 170 175180 Phe Asn Arg Glu Pro Ala Ile Val Arg Phe Phe Ser Arg Phe Thr 185 190195 Glu Val Arg Glu Phe Ala Ile Val Pro Leu His Ala Ala Pro Gly 200 205210 Asp Ala Val Ala Glu Ile Asp Ala Leu Tyr Asp Val Tyr Leu Asp 215 220225 Val Gln Glu Lys Trp Gly Leu Glu Asp Val Met Leu Met Gly Asp 230 235240 Phe Asn Ala Gly Cys Ser Tyr Val Arg Pro Ser Gln Trp Ser Ser 245 250255 Ile Arg Leu Trp Thr Ser Pro Thr Phe Gln Trp Leu Ile Pro Asp 260 265270 Ser Ala Asp Thr Thr Ala Thr Pro Thr His Cys Ala Tyr Asp Arg 275 280285 Ile Val Val Ala Gly Met Leu Leu Arg Gly Ala Val Val Pro Asp 290 295300 Ser Ala Leu Pro Phe Asn Phe Gln Ala Ala Tyr Gly Leu Ser Asp 305 310315 Gln Leu Ala Gln Ala Ile Ser Asp His Tyr Pro Val Glu Val Met 320 325330 Leu Lys Ala Ala Pro Pro His Thr Ser Thr Ala 335 340 260 amino acidsAmino Acid Linear 14 Leu Lys Ile Ala Ala Phe Asn Ile Gln Thr Phe Gly GluThr Lys 1 5 10 15 Met Ser Asn Ala Thr Leu Val Ser Tyr Ile Val Gln IleLeu Ser 20 25 30 Arg Tyr Asp Ile Ala Leu Val Gln Glu Val Arg Asp Ser HisLeu 35 40 45 Thr Ala Val Gly Lys Leu Leu Asp Asn Leu Asn Gln Asp Ala Pro50 55 60 Asp Thr Tyr His Tyr Val Val Ser Glu Pro Leu Gly Arg Asn Ser 6570 75 Tyr Lys Glu Arg Tyr Leu Phe Val Tyr Arg Pro Asp Gln Val Ser 80 8590 Ala Val Asp Ser Tyr Tyr Tyr Asp Asp Gly Cys Glu Pro Cys Gly 95 100105 Asn Asp Thr Phe Asn Arg Glu Pro Ala Ile Val Arg Phe Phe Ser 110 115120 Arg Phe Thr Glu Val Arg Glu Phe Ala Ile Val Pro Leu His Ala 125 130135 Ala Pro Gly Asp Ala Val Ala Glu Ile Asp Ala Leu Tyr Asp Val 140 145150 Tyr Leu Asp Val Gln Glu Lys Trp Gly Leu Glu Asp Val Met Leu 155 160165 Met Gly Asp Phe Asn Ala Gly Cys Ser Tyr Val Arg Pro Ser Gln 170 175180 Trp Ser Ser Ile Arg Leu Trp Thr Ser Pro Thr Phe Gln Trp Leu 185 190195 Ile Pro Asp Ser Ala Asp Thr Thr Ala Thr Pro Thr His Cys Ala 200 205210 Tyr Asp Arg Ile Val Val Ala Gly Met Leu Leu Arg Gly Ala Val 215 220225 Val Pro Asp Ser Ala Leu Pro Phe Asn Phe Gln Ala Ala Tyr Gly 230 235240 Leu Ser Asp Gln Leu Ala Gln Ala Ile Ser Asp His Tyr Pro Val 245 250255 Glu Val Met Leu Lys 260 260 amino acids Amino Acid Linear 15 Leu LysIle Ala Ala Phe Asn Ile Arg Thr Phe Gly Glu Thr Lys 1 5 10 15 Met SerAsn Ala Thr Leu Ala Ser Tyr Ile Val Arg Ile Val Arg 20 25 30 Arg Tyr AspIle Val Leu Ile Glu Gln Val Arg Asp Ser His Leu 35 40 45 Val Ala Val GlyLys Leu Leu Asp Tyr Leu Asn Gln Asp Asp Pro 50 55 60 Asn Thr Tyr His TyrVal Val Ser Glu Pro Leu Gly Arg Asn Ser 65 70 75 Tyr Lys Glu Arg Tyr LeuPhe Leu Phe Arg Pro Asn Lys Val Ser 80 85 90 Val Leu Asp Thr Tyr Gln TyrAsp Asp Gly Cys Glu Ser Cys Gly 95 100 105 Asn Asp Ser Phe Ser Arg GluPro Ala Val Val Lys Phe Ser Ser 110 115 120 His Ser Thr Lys Val Lys GluPhe Ala Ile Val Ala Leu His Ser 125 130 135 Ala Pro Ser Asp Ala Val AlaGlu Ile Asn Ser Leu Tyr Asp Val 140 145 150 Tyr Leu Asp Val Gln Gln LysTrp His Leu Asn Asp Val Met Leu 155 160 165 Met Gly Asp Phe Asn Ala AspCys Ser Tyr Val Thr Ser Ser Gln 170 175 180 Trp Ser Ser Ile Arg Leu ArgThr Ser Ser Thr Phe Gln Trp Leu 185 190 195 Ile Pro Asp Ser Ala Asp ThrThr Ala Thr Ser Thr Asn Cys Ala 200 205 210 Tyr Asp Arg Ile Val Val AlaGly Ser Leu Leu Gln Ser Ser Val 215 220 225 Val Gly Pro Ser Ala Ala ProPhe Asp Phe Gln Ala Ala Tyr Gly 230 235 240 Leu Ser Asn Glu Met Ala LeuAla Ile Ser Asp His Tyr Pro Val 245 250 255 Glu Val Thr Leu Thr 260 664base pairs Nucleic Acid Double Linear 16 TTCGAGCTCG CCCGACATTGATTATTGACT AGAGTCGACA GCTGTGGAAT 50 GTGTGTCAGT TAGGGTGTGG AAAGTCCCCAGGCTCCCCAG CAGGCAGAAG 100 TATGCAAAGC ATGCATCTCA ATTAGTCAGC AACCAGGTGTGGAAAGTCCC 150 CAGGCTCCCC AGCAGGCAGA AGTATGCAAA GCATGCATCT CAATTAGTCA200 GCAACCATAG TCCCGCCCCT AACTCCGCCC ATCCCGCCCC TAACTCCGCC 250CAGTTCCGCC CATTCTCCGC CCCATGGCTG ACTAATTTTT TTTATTTATG 300 CAGAGGCCGAGGCCGCCTCG GCCTCTGAGC TATTCCAGAA GTAGTGAGGA 350 GGCTTTTTTG GAGGCCTAGGCTTTTGCAAA AAGCTTATCG GGCCGGGAAC 400 GGTGCATTGG AACGCGGATT CCCCGTGCCAAGAGTGACGT AAGTACCGCC 450 TATAGAGTCT ATAGGCCCAC CCCCTTGGCT TCGTTAGAACGCGGCTACAA 500 TTAATACATA ACCTTATGTA TCATACACAT ACGATTTAGG TGACACTATA550 GAATAACATC CACTTTGCCT TTCTCTCCAC AGGTGTCCAC TCCCAGGTCC 600AACTGCACCT CGGTTCTAAG CTTGGGCTGC AGGTCGCCGT GAATTTAAGG 650 GACGCTGTGAAGCA 664 664 base pairs Nucleic Acid Double Linear 17 TTCGAGCTCGCCCGACATTG ATTATTGACT AGAGTCGACA GCTGTGGAAT 50 GTGTGTCAGT TAGGGTGTGGAAAGTCCCCA GGCTCCCCAG CAGGCAGAAG 100 TATGCAAAGC ATGCATCTCA ATTAGTCAGCAACCAGGTGT GGAAAGTCCC 150 CAGGCTCCCC AGCAGGCAGA AGTATGCAAA GCATGCATCTCAATTAGTCA 200 GCAACCATAG TCCCGCCCCT AACTCCGCCC ATCCCGCCCC TAACTCCGCC250 CAGTTCCGCC CATTCTCCGC CCCATGGCTG ACTAATTTTT TTTATTTATG 300CAGAGGCCGA GGCCGCCTCG GCCTCTGAGC TATTCCAGAA GTAGTGAGGA 350 GGCTTTTTTGGAGGCCTAGG CTTTTGCAAA AAGCTTATCC GGCCGGGAAC 400 GGTGCATTGG AACGCGGATTCCCCGTGCCA AGAGTCAGGT AAGTACCGCC 450 TATAGAGTCT ATAGGCCCAC CCCCTTGGCTTCGTTAGAAC GCGGCTACAA 500 TTAATACATA ACCTTATGTA TCATACACAT ACGATTTAGGTGACACTATA 550 GAATAACATC CACTTTGCCT TTCTCTCCAC AGGTGTCCAC TCCCAGGTCC600 AACTGCACCT CGGTTCTAAG CTTGGGCTGC AGGTCGCCGT GAATTTAAGG 650GACGCTGTGA AGCA 664 640 base pairs Nucleic Acid Double Linear 18TTCGAGCTCG CCCGACATTG ATTATTGACT AGAGTCGACA GCTGTGGAAT 50 GTGTGTCAGTTAGGGTGTGG AAAGTCCCCA GGCTCCCCAG CAGGCAGAAG 100 TATGCAAAGC ATGCATCTCAATTAGTCAGC AACCAGGTGT GGAAAGTCCC 150 CAGGCTCCCC AGCAGGCAGA AGTATGCAAAGCATGCATCT CAATTAGTCA 200 GCAACCATAG TCCCGCCCCT AACTCCGCCC ATCCCGCCCCTAACTCCGCC 250 CAGTTCCGCC CATTCTCCGC CCCATGGCTG ACTAATTTTT TTTATTTATG300 CAGAGGCCGA GGCCGCCTCG GCCTCTGAGC TATTCCAGAA GTAGTGAGGA 350GGCTTTTTTG GAGGCCTAGG CTTTTGCAAA AAGCTTATCC GGCCGGGAAC 400 GGTGCATTGGAACGCGGATT CCCCGTGCCA AGAGTCAGGT AAGTACCGCC 450 TATAGAGTCT ATAGGCCCACCCCCTTGGCT TCGTTAGAAC GCGGCTACAA 500 TTAATACATA ACCTTTTGGA TCCTATAGACTGACATCCAC TTTGCCTTTC 550 TCTCCACAGG TGTCCACTCC CAGGTCCAAC TGCACCTCGGTTCGAAGCTT 600 GGGCTGCAGG TCGCCGTGAA TTTAAGGGAC GCTGTGAAGC 640 646 basepairs Nucleic Acid Double Linear 19 TTCGAGCTCG CCCGACATTG ATTATTGACTAGAGTCGACA GCTGTGGAAT 50 GTGTGTCAGT TAGGGTGTGG AAAGTCCCCA GGCTCCCCAGCAGGCAGAAG 100 TATGCAAAGC ATGCATCTCA ATTAGTCAGC AACCAGGTGT GGAAAGTCCC150 CAGGCTCCCC AGCAGGCAGA AGTATGCAAA GCATGCATCT CAATTAGTCA 200GCAACCATAG TCCCGCCCCT AACTCCGCCC ATCCCGCCCC TAACTCCGCC 250 CAGTTCCGCCCATTCTCCGC CCCATGGCTG ACTAATTTTT TTTATTTATG 300 CAGAGGCCGA GGCCGCCTCGGCCTCTGAGC TATTCCAGAA GTAGTGAGGA 350 GGCTTTTTTG GAGGCCTAGG CTTTTGCAAAAAGCTTATCC GGCCGGGAAC 400 GGTGCATTGG AACGCGGATT CCCCGTGCCA AGAGTCAGGTAAGTACCGCC 450 TATAGAGTCT ATAGGCCCAC CCCCTTGGCT TCGTTAGAAC GCGGCTACAA500 TTAATACATA ACCTTTTGGA TCCTACTAAC TACTGACTTA TTCTTTTCCT 550TTCTCTCCAC AGGTGTCCAC TCCCAGGTCC AACTGCACCT CGGTTCGCGA 600 AGCTTGGGCTGCAGGTCGCC GTGAATTTAA GGGACGCTGT GAAGCA 646 645 base pairs Nucleic AcidDouble Linear 20 TTCGAGCTCG CCCGACATTG ATTATTGACT AGAGTCGACA GCTGTGGAAT50 GTGTGTCAGT TAGGGTGTGG AAAGTCCCCA GGCTCCCCAG CAGGCAGAAG 100 TATGCAAAGCATGCATCTCA ATTAGTCAGC AACCAGGTGT GGAAAGTCCC 150 CAGGCTCCCC AGCAGGCAGAAGTATGCAAA GCATGCATCT CAATTAGTCA 200 GCAACCATAG TCCCGCCCCT AACTCCGCCCATCCCGCCCC TAACTCCGCC 250 CAGTTCCGCC CATTCTCCGC CCCATGGCTG ACTAATTTTTTTTATTTATG 300 CAGAGGCCGA GGCCGCCTCG GCCTCTGAGC TATTCCAGAA GTAGTGAGGA350 GGCTTTTTTG GAGGCCTAGG CTTTTGCAAA AAGCTTATCC GGCCGGGAAC 400GGTGCATTGG AACGCGGATT CCCCGTGCCA AGAGTCAGGT AAGTACCGCC 450 TATAGAGTCTATAGGCCCAC CCCCTTGGCT TCGTTAGAAC GCGGCTACAA 500 TTAATACATA ACCTTTTGGATCCTACTGAC ACTGACATCC ACTTTTTCTT 550 TTTCTCCACA GGTGTCCACT CCCAGGTCCAACTGCACCTC GGTTCGCGAA 600 GCTTGGGCTG CAGGTCGCCG TGAATTTAAG GGACGCTGTGAAGCA 645 81 base pairs Nucleic Acid Single Linear 21 TGACGTAAGTACATGTATCA TACACATACG ATTTAGGTGA CACTATAGAA 50 TAACATCCAC TTTGCCTTTCTCTCCACAGG T 81 81 base pairs Nucleic Acid Single Linear 22 TCAGGTAAGTACATGTATCA TACACATACG ATTTAGGTGA CACTATAGAA 50 TAACATCCAC TTTGCCTTTCTCTCCACAGG T 81 58 base pairs Nucleic Acid Single Linear 23 TCAGGTAAGTACTTGGATCC TATAGACTGA CATCCACTTT GCCTTTCTCT 50 CCACAGGT 58 61 base pairsNucleic Acid Single Linear 24 TCAGGTAAGT ACTTGGATCC TACTAACTACTGACTTATTC TTTTCCTTTC 50 TCTCCACAGG T 61 60 base pairs Nucleic AcidSingle Linear 25 TCAGGTAAGT ACTTGGATCC TACTGACACT GACATCCACT TTTTCTTTTT50 CTCCACAGGT 60

1. Human DNase unaccompanied by associated native glycosylation.
 2. ADNA isolate encoding human DNase.
 3. The isolate of claim 2 wherein theisolate is free of DNase introns.
 4. The isolate of claim 2 wherein theisolate is free of genomic DNA which encodes another polypeptide fromthe source of the DNA.
 5. The isolate of claim 2, wherein the DNAencodes a polypeptide having the amino sequence shown in FIG.
 1. 6. Arecombinant expression vector comprising DNA encoding human DNase.
 7. Acomposition comprising a cell transformed with the recombinantexpression vector of claim
 6. 8. The composition of claim 7 wherein thecell is a mammalian cell.
 9. A process for producing DNase whichcomprises transforming a host cell with nucleic acid encoding DNase,culturing the transformed cell and recovering DNase from the culture.10. The process according to claim 9 wherein the DNase is recovered fromthe culture medium of the host cell.
 11. The process according to claim10 wherein the host cell is a eukaryotic cell.
 12. The process of claim9 wherein the DNase is human DNase.
 13. The process of claim 12 whereinthe eukaryotic cell is a human embryonic kidney cell line.
 14. Theprocess of claim 12 wherein the nucleic acid encodes a human DNasepreprotein.
 15. The process of claim 14 wherein the preprotein is humanpreDNase.
 16. The process of claim 12 wherein the human DNase issecreted into the culture medium.
 17. The process of claim 12 whereinthe human DNase is unglycosylated.
 18. A pharmaceutical preparationuseful for enzymatic treatment comprising a therapeutically effectiveamount of human DNase and a pharmaceutically acceptable carrier.
 19. Thepreparation of claim 18 which is sterile.
 20. The preparation of claim18 wherein the DNase is entirely free of proteases.
 21. The preparationof claim 20 in the form of an aerosol.
 22. The preparation of claim 18wherein the DNase has the sequence of the mature DNase depicted inFIG.
 1. 23. A polynucleotide probe containing at least about 10 baseswhich is capable of hybridizing under stringent conditions to the humanDNase gene.
 24. A conjugate of human DNase and a nonproteinaceouspolymer.
 25. The conjugate of claim 24 wherein the polymer is surgicaltubing selected from the group of catheters and drainage conduits. 26.The conjugate of claim 24 which is water soluble.
 27. The conjugate ofclaim 24 wherein the polymer is a polyoxyalkylene or polyalkyleneglycol.
 28. A method for the treatment of a patient having anaccumulation of purulent material comprising administering pure DNasefree of proteases to the patient in an amount therapeutically effectiveto reduce the visco-elasticity of the material.
 29. A method forenhancing the activity of antibiotics comprising administering to apatient a therapeutically effective amount of an antibiotic and atherapeutically effective amount of DNase.
 30. A method for maintainingthe flow of fluid in conduits communicating with a patient's body cavitycomprising contacting the interior of the conduit with DNase.
 31. Themethod of claim 30 wherein the DNase is covalently bound to the conduit.32. The method of claim 30 wherein a solution of the DNase is passedthrough the conduit into the body cavity.
 33. A method for purifyingpharmaceutical preparations so as to be free of contaminant DNAcomprising contacting the preparation with DNase under conditions fordegrading the contaminant DNA to oligonucleotides and removing theoligonucleotides and DNase from the preparation.
 34. The method of claim33 wherein the DNase is immobilized on a water insoluble support.
 35. Amethod for the treatment of a patient having cystic fibrosis comprisingadministering to such patient a therapeutically effective dose of pureDNase free of proteases.